Manufacturing Methods for Producing Anti-TNF Antibody Compositions

ABSTRACT

The present invention relates to methods of manufacture for producing anti-TNF antibodies, e.g., the anti-TNF antibody a recombinant anti-TNF antibody having a heavy chain (HC) comprising amino acid sequence SEQ ID NO:36 and a light chain (LC) comprising amino acid sequence SEQ ID NO:37 and compositions comprising the recombinant anti-TNF antibody.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “JBI6055USPSP1SEQLIST.TXT” creation date of Feb. 5, 2019 andhaving a size of 21,508 bytes. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of manufacture for producinganti-TNF antibodies, e.g., the anti-TNF antibody a recombinant anti-TNFantibody having a heavy chain (HC) comprising amino acid sequence SEQ IDNO:36 and a light chain (LC) comprising amino acid sequence SEQ ID NO:37and compositions comprising the recombinant anti-TNF antibody.

BACKGROUND OF THE INVENTION

TNF alpha is a soluble homotrimer of 17 kD protein subunits. Amembrane-bound 26 kD precursor form of TNF also exists.

Cells other than monocytes or macrophages also produce TNF alpha. Forexample, human non-monocytic tumor cell lines produce TNF alpha and CD4+and CD8+ peripheral blood T lymphocytes and some cultured T and B celllines also produce TNF alpha.

TNF alpha causes pro-inflammatory actions which result in tissue injury,such as degradation of cartilage and bone, induction of adhesionmolecules, inducing procoagulant activity on vascular endothelial cells,increasing the adherence of neutrophils and lymphocytes, and stimulatingthe release of platelet activating factor from macrophages, neutrophilsand vascular endothelial cells.

TNF alpha has been associated with infections, immune disorders,neoplastic pathologies, autoimmune pathologies and graft-versus-hostpathologies. The association of TNF alpha with cancer and infectiouspathologies is often related to the host's catabolic state. Cancerpatients suffer from weight loss, usually associated with anorexia.

The extensive wasting which is associated with cancer, and otherdiseases, is known as “cachexia”. Cachexia includes progressive weightloss, anorexia, and persistent erosion of lean body mass in response toa malignant growth. The cachectic state causes much cancer morbidity andmortality. There is evidence that TNF alpha is involved in cachexia incancer, infectious pathology, and other catabolic states.

TNF alpha is believed to play a central role in gram-negative sepsis andendotoxic shock, including fever, malaise, anorexia, and cachexia.Endotoxin strongly activates monocyte/macrophage production andsecretion of TNF alpha and other cytokines. TNF alpha and othermonocyte-derived cytokines mediate the metabolic and neurohormonalresponses to endotoxin. Endotoxin administration to human volunteersproduces acute illness with flu-like symptoms including fever,tachycardia, increased metabolic rate and stress hormone release.Circulating TNF alpha increases in patients suffering from Gram-negativesepsis.

Thus, TNF alpha has been implicated in inflammatory diseases, autoimmunediseases, viral, bacterial and parasitic infections, malignancies,and/or neurodegenerative diseases and is a useful target for specificbiological therapy in diseases, such as rheumatoid arthritis and Crohn'sdisease. Beneficial effects in open-label trials with monoclonalantibodies to TNF alpha have been reported with suppression ofinflammation and with successful retreatment after relapse in rheumatoidarthritis and in Crohn's disease. Beneficial results in a randomized,double-blind, placebo-controlled trials have also been reported inrheumatoid arthritis with suppression of inflammation.

Neutralizing antisera or mAbs to TNF have been shown in mammals otherthan man to abrogate adverse physiological changes and prevent deathafter lethal challenge in experimental endotoxemia and bacteremia. Thiseffect has been demonstrated, e.g., in rodent lethality assays and inprimate pathology model systems.

Putative receptor binding loci of hTNF has been disclosed and thereceptor binding loci of TNF alpha as consisting of amino acids 11-13,37-42, 49-57 and 155-157 of TNF have been disclosed.

Non-human mammalian, chimeric, polyclonal (e.g., anti-sera) and/ormonoclonal antibodies (Mabs) and fragments (e.g., proteolytic digestionor fusion protein products thereof) are potential therapeutic agentsthat are being investigated in some cases to attempt to treat certaindiseases. However, such antibodies or fragments can elicit an immuneresponse when administered to humans. Such an immune response can resultin an immune complex-mediated clearance of the antibodies or fragmentsfrom the circulation, and make repeated administration unsuitable fortherapy, thereby reducing the therapeutic benefit to the patient andlimiting the re-administration of the antibody or fragment. For example,repeated administration of antibodies or fragments comprising non-humanportions can lead to serum sickness and/or anaphylaxis. In order toavoid these and other problems, a number of approaches have been takento reduce the immunogenicity of such antibodies and portions thereof,including chimerization and humanization, as well known in the art.These and other approaches, however, still can result in antibodies orfragments having some immunogenicity, low affinity, low avidity, or withproblems in cell culture, scale up, production, and/or low yields. Thus,such antibodies or fragments can be less than ideally suited formanufacture or use as therapeutic proteins.

Accordingly, there is a need to provide anti-TNF antibodies or fragmentsthereof for use as therapeutics for treatment of diseases mediated byTNF alpha.

SUMMARY OF THE INVENTION

The embodiments of the invention are defined, respectively, by theindependent and dependent claims appended hereto, which for the sake ofbrevity are incorporated by reference herein. Other embodiments,features, and advantages of the various aspects of the invention areapparent from the detailed description below taken in conjunction withthe appended drawing figures.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells).

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies comprises totalneutral oligosaccharide species >99.0% and total charged oligosaccharidespecies <1.0%.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies further comprisesindividual neutral oligosaccharide species G0F >60.0%, G1F <20.0%, andG2F <5.0%.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have no disialylated glycan species as determined byHigh Performance Liquid Chromatography (HPLC) or Reduced Mass Analysis(RMA).

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies are a follow-on biologic.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies comprises totalneutral oligosaccharide species >99.0% and total charged oligosaccharidespecies <1.0%, and wherein the anti-TNF antibodies have a longerhalf-life or increased antibody-dependent cell-mediated cytotoxicity(ADCC) compared to anti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies further comprisesindividual neutral oligosaccharide species G0F >60.0%, G1F <20.0%, andG2F <5.0%, and wherein the anti-TNF antibodies have a longer half-lifeor increased antibody-dependent cell-mediated cytotoxicity (ADCC)compared to anti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have no disialylated glycan species as determined byHigh Performance Liquid Chromatography (HPLC) or Reduced Mass Analysis(RMA), and wherein the anti-TNF antibodies have a longer half-life orincreased antibody-dependent cell-mediated cytotoxicity (ADCC) comparedto anti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells and the anti-TNF antibodiesare a follow-on biologic.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells and the anti-TNF antibodiesare a follow-on biologic.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies comprises totalneutral oligosaccharide species >99.0% and total charged oligosaccharidespecies <1.0%, and wherein the anti-TNF antibodies have a longerhalf-life or increased antibody-dependent cell-mediated cytotoxicity(ADCC) compared to anti-TNF antibodies expressed in Sp2/0 cells and theanti-TNF antibodies are a follow-on biologic.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies further comprisesindividual neutral oligosaccharide species G0F >60.0%, G1F <20.0%, andG2F <5.0%, and wherein the anti-TNF antibodies have a longer half-lifeor increased antibody-dependent cell-mediated cytotoxicity (ADCC)compared to anti-TNF antibodies expressed in Sp2/0 cells and theanti-TNF antibodies are a follow-on biologic.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have no disialylated glycan species as determined byHigh Performance Liquid Chromatography (HPLC) or Reduced Mass Analysis(RMA), and wherein the anti-TNF antibodies have a longer half-life orincreased antibody-dependent cell-mediated cytotoxicity (ADCC) comparedto anti-TNF antibodies expressed in Sp2/0 cells and the anti-TNFantibodies are a follow-on biologic.

In certain embodiments, the present invention provides anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells and the anti-TNF antibodiesare a follow-on biologic.

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies.

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,wherein the oligosaccharide profile of the anti-TNF antibodies comprisestotal neutral oligosaccharide species >99.0% and total chargedoligosaccharide species <1.0%, and

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,wherein the oligosaccharide profile of the anti-TNF antibodies furthercomprises individual neutral oligosaccharide species G0F >60.0%, G1F<20.0%, and G2F <5.0%.

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,wherein the anti-TNF antibodies have no disialylated glycan species asdetermined by High Performance Liquid Chromatography (HPLC) or ReducedMass Analysis (RMA).

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,wherein the anti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,wherein the anti-TNF antibodies are a follow-on biologic.

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,wherein the oligosaccharide profile of the anti-TNF antibodies comprisestotal neutral oligosaccharide species >99.0% and total chargedoligosaccharide species <1.0%, and wherein the anti-TNF antibodies havea longer half-life or increased antibody-dependent cell-mediatedcytotoxicity (ADCC) compared to anti-TNF antibodies expressed in Sp2/0cells.

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,wherein the oligosaccharide profile of the anti-TNF antibodies furthercomprises individual neutral oligosaccharide species G0F >60.0%, G1F<20.0%, and G2F <5.0%, and wherein the anti-TNF antibodies have a longerhalf-life or increased antibody-dependent cell-mediated cytotoxicity(ADCC) compared to anti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,wherein the anti-TNF antibodies have no disialylated glycan species asdetermined by High Performance Liquid Chromatography (HPLC) or ReducedMass Analysis (RMA), and wherein the anti-TNF antibodies have a longerhalf-life or increased antibody-dependent cell-mediated cytotoxicity(ADCC) compared to anti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,wherein the anti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides a method ofmanufacture for producing anti-TNF antibodies comprising: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are produced by a method of manufacturecomprising: a. culturing Chinese Hamster Ovary cells (CHO cells) withnucleotides encoding the anti-TNF antibodies; b. expressing the anti-TNFantibodies in the CHO cells; and, c. purifying the anti-TNF antibodies,and wherein the anti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells the anti-TNF antibodies area follow-on biologic.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells).

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies comprises totalneutral oligosaccharide species >99.0% and total charged oligosaccharidespecies <1.0%.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies further comprisesindividual neutral oligosaccharide species G0F >60.0%, G1F <20.0%, andG2F <5.0%.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have no disialylated glycan species as determined byHigh Performance Liquid Chromatography (HPLC).

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies are a follow-on biologic.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies comprises totalneutral oligosaccharide species >99.0% and total charged oligosaccharidespecies <1.0%, and wherein the anti-TNF antibodies have a longerhalf-life or increased antibody-dependent cell-mediated cytotoxicity(ADCC) compared to anti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies further comprisesindividual neutral oligosaccharide species G0F >60.0%, G1F <20.0%, andG2F <5.0%, and wherein the anti-TNF antibodies have a longer half-lifeor increased antibody-dependent cell-mediated cytotoxicity (ADCC)compared to anti-TNF antibodies expressed in Sp2/0 cells.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have no disialylated glycan species as determined byHigh Performance Liquid Chromatography (HPLC), and wherein the anti-TNFantibodies have a longer half-life or increased antibody-dependentcell-mediated cytotoxicity (ADCC) compared to anti-TNF antibodiesexpressed in Sp2/0 cells.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells, and wherein the anti-TNFantibodies have a longer half-life or increased antibody-dependentcell-mediated cytotoxicity (ADCC) compared to anti-TNF antibodiesexpressed in Sp2/0 cells.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies comprises totalneutral oligosaccharide species >99.0% and total charged oligosaccharidespecies <1.0%, and wherein the anti-TNF antibodies have a longerhalf-life or increased antibody-dependent cell-mediated cytotoxicity(ADCC) compared to anti-TNF antibodies expressed in Sp2/0 cells and theanti-TNF antibodies are a follow-on biologic.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theoligosaccharide profile of the anti-TNF antibodies further comprisesindividual neutral oligosaccharide species G0F >60.0%, G1F <20.0%, andG2F <5.0%, and wherein the anti-TNF antibodies have a longer half-lifeor increased antibody-dependent cell-mediated cytotoxicity (ADCC)compared to anti-TNF antibodies expressed in Sp2/0 cells and theanti-TNF antibodies are a follow-on biologic.

In certain embodiments, the present invention provides a compositioncomprising anti-TNF antibodies: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells), and wherein theanti-TNF antibodies have no disialylated glycan species as determined byHigh Performance Liquid Chromatography (HPLC), and wherein the anti-TNFantibodies have a longer half-life or increased antibody-dependentcell-mediated cytotoxicity (ADCC) compared to anti-TNF antibodiesexpressed in Sp2/0 cells and the anti-TNF antibodies are a follow-onbiologic.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a graphical representation showing an assay for ability ofTNV mAbs in hybridoma cell supernatants to inhibit TNFα binding torecombinant TNF receptor. Varying amounts of hybridoma cell supernatantscontaining known amounts of TNV mAb were preincubated with a fixedconcentration (5 ng/ml) of ¹²⁵I-labeled TNFα. The mixture wastransferred to 96-well Optiplates that had been previously coated withp55-sf2, a recombinant TNF receptor/IgG fusion protein. The amount ofTNFα that bound to the p55 receptor in the presence of the mAbs wasdetermined after washing away the unbound material and counting using agamma counter. Although eight TNV mAb samples were tested in theseexperiments, for simplicity three of the mAbs that were shown by DNAsequence analyses to be identical to one of the other TNV mAbs are notshown here. Each sample was tested in duplicate. The results shown arerepresentative of two independent experiments.

FIG. 2A-B shows DNA sequences of the TNV mAb heavy chain variableregions. The germline gene shown is the DP-46 gene. ‘TNVs’ indicatesthat the sequence shown is the sequence of TNV14, TNV15, TNV148, andTNV196. The first three nucleotides in the TNV sequence define thetranslation initiation Met codon. Dots in the TNV mAb gene sequencesindicate the nucleotide is the same as in the germline sequence. Thefirst 19 nucleotides (underlined) of the TNV sequences correspond to theoligonucleotide used to PCR-amplify the variable region. An amino acidtranslation (single letter abbreviations) starting with the mature mAbis shown only for the germline gene. The three CDR domains in thegermline amino acid translation are marked in bold and underlined. Lineslabeled TNV148(B) indicate that the sequence shown pertains to bothTNV148 and TNV148B. Gaps in the germline DNA sequence (CDR3) were due tothe sequence not being known or not existing in the germline gene at thetime. The TNV mAb heavy chains use the J6 joining region.

FIG. 3 shows DNA sequences of the TNV mAb light chain variable regions.The germline gene shown is a representative member of the Vg/38K familyof human kappa germline variable region genes. Dots in the TNV mAb genesequences indicate the nucleotide is the same as in the germlinesequence. The first 16 nucleotides (underlined) of the TNV sequencescorrespond to the oligonucleotide used to PCR-amplify the variableregion. An amino acid translation of the mature mAb (single letterabbreviations) is shown only for the germline gene. The three CDRdomains in the germline amino acid translation are marked in bold andunderlined. Lines labeled TNV148(B) indicate that the sequence shownpertains to both TNV148 and TNV148B. Gaps in the germline DNA sequence(CDR3) are due to the sequence not being known or not existing in thegermline gene. The TNV mAb light chains use the J3 joining sequence.

FIG. 4 shows deduced amino acid sequences of the TNV mAb heavy chainvariable regions. The amino acid sequences shown (single letterabbreviations) were deduced from DNA sequence determined from bothuncloned PCR products and cloned PCR products. The amino sequences areshown partitioned into the secretory signal sequence (signal), framework(FW), and complementarity determining region (CDR) domains. The aminoacid sequence for the DP-46 germline gene is shown on the top line foreach domain. Dots indicate that the amino acid in the TNV mAb isidentical to the germline gene. TNV148(B) indicates that the sequenceshown pertains to both TNV148 and TNV148B. ‘TNVs’ indicates that thesequence shown pertains to all TNV mAbs unless a different sequence isshown. Dashes in the germline sequence (CDR3) indicate that thesequences are not known or do not exist in the germline gene.

FIG. 5 shows deduced amino acid sequences of the TNV mAb light chainvariable regions. The amino acid sequences shown (single letterabbreviations) were deduced from DNA sequence determined from bothuncloned PCR products and cloned PCR products. The amino sequences areshown partitioned into the secretory signal sequence (signal), framework(FW), and complementarity determining region (CDR) domains. The aminoacid sequence for the Vg/38K-type light chain germline gene is shown onthe top line for each domain. Dots indicate that the amino acid in theTNV mAb is identical to the germline gene. TNV148 (B) indicates that thesequence shown pertains to both TNV148 and TNV148B. ‘All’ indicates thatthe sequence shown pertains to TNV14, TNV15, TNV148, TNV148B, andTNV186.

FIG. 6 shows schematic illustrations of the heavy and light chainexpression plasmids used to make the rTNV148B-expressing C466 cells.p1783 is the heavy chain plasmid and p1776 is the light chain plasmid.The rTNV148B variable and constant region coding domains are shown asblack boxes. The immunoglobulin enhancers in the J-C introns are shownas gray boxes. Relevant restriction sites are shown. The plasmids areshown oriented such that transcription of the Ab genes proceeds in aclockwise direction. Plasmid p1783 is 19.53 kb in length and plasmidp1776 is 15.06 kb in length. The complete nucleotide sequences of bothplasmids are known. The variable region coding sequence in p1783 can beeasily replaced with another heavy chain variable region sequence byreplacing the BsiWI/BstBI restriction fragment. The variable regioncoding sequence in p1776 can be replaced with another variable regionsequence by replacing the SalI/AflII restriction fragment.

FIG. 7 shows graphical representation of growth curve analyses of fiverTNV148B-producing cell lines. Cultures were initiated on day 0 byseeding cells into T75 flasks in I5Q+MHX media to have a viable celldensity of 1.0×10⁵ cells/ml in a 30 ml volume. The cell cultures usedfor these studies had been in continuous culture since transfections andsubclonings were performed. On subsequent days, cells in the T flaskswere thoroughly resuspended and a 0.3 ml aliquot of the culture wasremoved. The growth curve studies were terminated when cell countsdropped below 1.5×10⁵ cells/ml. The number of live cells in the aliquotwas determined by trypan blue exclusion and the remainder of the aliquotstored for later mAb concentration determination. An ELISA for human IgGwas performed on all sample aliquots at the same time.

FIG. 8 shows a graphical representation of the comparison of cell growthrates in the presence of varying concentrations of MHX selection. Cellsubclones C466A and C466B were thawed into MHX-free media (IMDM, 5% FBS,2 mM glutamine) and cultured for two additional days. Both cell cultureswere then divided into three cultures that contained either no MHX,0.2×MHX, or 1×MHX. One day later, fresh T75 flasks were seeded with thecultures at a starting density of 1×10⁵ cells/ml and cells counted at 24hour intervals for one week. Doubling times during the first 5 days werecalculated using the formula in SOP PD32.025 and are shown above thebars.

FIG. 9 shows graphical representations of the stability of mAbproduction over time from two rTNV148B-producing cell lines. Cellsubclones that had been in continuous culture since performingtransfections and subclonings were used to start long-term serialcultures in 24-well culture dishes. Cells were cultured in I5Q mediawith and without MHX selection. Cells were continually passaged bysplitting the cultures every 4 to 6 days to maintain new viable cultureswhile previous cultures were allowed to go spent. Aliquots of spent cellsupernatant were collected shortly after cultures were spent and storeduntil the mAb concentrations were determined. An ELISA for human IgG wasperformed on all sample aliquots at the same time.

FIG. 10 shows arthritis mouse model mice Tg 197 weight changes inresponse to anti-TNF antibodies of the present invention as compared tocontrols in Example 4. At approximately 4 weeks of age the Tg197 studymice were assigned, based on gender and body weight, to one of 9treatment groups and treated with a single intraperitoneal bolus dose ofDulbecco's PBS (D-PBS) or an anti-TNF antibody of the present invention(TNV14, TNV148 or TNV196) at either 1 mg/kg or 10 mg/kg. When theweights were analyzed as a change from pre-dose, the animals treatedwith 10 mg/kg cA2 showed consistently higher weight gain than theD-PBS-treated animals throughout the study. This weight gain wassignificant at weeks 3-7. The animals treated with 10 mg/kg TNV148 alsoachieved significant weight gain at week 7 of the study.

FIG. 11A-C represent the progression of disease severity based on thearthritic index as presented in Example 4. The 10 mg/kg cA2-treatedgroup's arthritic index was lower than the D-PBS control group startingat week 3 and continuing throughout the remainder of the study (week 7).The animals treated with 1 mg/kg TNV14 and the animals treated with 1mg/kg cA2 failed to show significant reduction in AI after week 3 whencompared to the D-PBS-treated Group. There were no significantdifferences between the 10 mg/kg treatment groups when each was comparedto the others of similar dose (10 mg/kg cA2 compared to 10 mg/kg TNV14,148 and 196). When the 1 mg/kg treatment groups were compared, the 1mg/kg TNV148 showed a significantly lower AI than 1 mg/kg cA2 at 3, 4and 7 weeks. The 1 mg/kg TNV148 was also significantly lower than the 1mg/kg TNV14-treated Group at 3 and 4 weeks. Although TNV196 showedsignificant reduction in AI up to week 6 of the study (when compared tothe D-PBS-treated Group), TNV148 was the only 1 mg/kg treatment thatremained significant at the conclusion of the study.

FIG. 12 shows arthritis mouse model mice Tg 197 weight changes inresponse to anti-TNF antibodies of the present invention as compared tocontrols in Example 5. At approximately 4 weeks of age the Tg197 studymice were assigned, based on body weight, to one of 8 treatment groupsand treated with a intraperitoneal bolus dose of control article (D-PBS)or antibody (TNV14, TNV148) at 3 mg/kg (week 0). Injections wererepeated in all animals at weeks 1, 2, 3, and 4. Groups 1-6 wereevaluated for test article efficacy. Serum samples, obtained fromanimals in Groups 7 and 8 were evaluated for immune response inductivelyand pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3 and 4.

FIG. 13A-C are graphs representing the progression of disease severityin Example 5 based on the arthritic index. The 10 mg/kg cA2-treatedgroup's arthritic index was significantly lower than the D-PBS controlgroup starting at week 2 and continuing throughout the remainder of thestudy (week 5). The animals treated with 1 mg/kg or 3 mg/kg of cA2 andthe animals treated with 3 mg/kg TNV14 failed to achieve any significantreduction in AI at any time throughout the study when compared to thed-PBS control group. The animals treated with 3 mg/kg TNV148 showed asignificant reduction when compared to the d-PBS-treated group startingat week 3 and continuing through week 5. The 10 mg/kg cA2-treatedanimals showed a significant reduction in AI when compared to both thelower doses (1 mg/kg and 3 mg/kg) of cA2 at weeks 4 and 5 of the studyand was also significantly lower than the TNV14-treated animals at weeks3-5. Although there appeared to be no significant differences betweenany of the 3 mg/kg treatment groups, the AI for the animals treated with3 mg/kg TNV14 were significantly higher at some time points than the 10mg/kg whereas the animals treated with TNV148 were not significantlydifferent from the animals treated with 10 mg/kg of cA2.

FIG. 14 shows arthritis mouse model mice Tg 197 weight changes inresponse to anti-TNF antibodies of the present invention as compared tocontrols in Example 6. At approximately 4 weeks of age the Tg197 studymice were assigned, based on gender and body weight, to one of 6treatment groups and treated with a single intraperitoneal bolus dose ofantibody (cA2, or TNV148) at either 3 mg/kg or 5 mg/kg. This studyutilized the D-PBS and 10 mg/kg cA2 control Groups.

FIG. 15 represents the progression of disease severity based on thearthritic index as presented in Example 6. All treatment groups showedsome protection at the earlier time points, with the 5 mg/kg cA2 and the5 mg/kg TNV148 showing significant reductions in AI at weeks 1-3 and alltreatment groups showing a significant reduction at week 2. Later in thestudy the animals treated with 5 mg/kg cA2 showed some protection, withsignificant reductions at weeks 4, 6 and 7. The low dose (3 mg/kg) ofboth the cA2 and the TNV148 showed significant reductions at 6 and alltreatment groups showed significant reductions at week 7. None of thetreatment groups were able to maintain a significant reduction at theconclusion of the study (week 8). There were no significant differencesbetween any of the treatment groups (excluding the saline control group)at any time point.

FIG. 16 shows arthritis mouse model mice Tg 197 weight changes inresponse to anti-TNF antibodies of the present invention as compared tocontrols in Example 7. To compare the efficacy of a singleintraperitoneal dose of TNV148 (derived from hybridoma cells) andrTNV148B (derived from transfected cells). At approximately 4 weeks ofage the Tg197 study mice were assigned, based on gender and body weight,to one of 9 treatment groups and treated with a single intraperitonealbolus dose of Dulbecco's PBS (D-PBS) or antibody (TNV148, rTNV148B) at 1mg/kg.

FIG. 17 represents the progression of disease severity based on thearthritic index as presented in Example 7. The 10 mg/kg cA2-treatedgroup's arthritic index was lower than the D-PBS control group startingat week 4 and continuing throughout the remainder of the study (week 8).Both of the TNV148-treated Groups and the 1 mg/kg cA2-treated Groupshowed a significant reduction in AI at week 4. Although a previousstudy (P-099-017) showed that TNV148 was slightly more effective atreducing the Arthritic Index following a single 1 mg/kg intraperitonealbolus, this study showed that the AI from both versions of the TNVantibody-treated groups was slightly higher. Although (with theexception of week 6) the 1 mg/kg cA2-treated Group was not significantlyincreased when compared to the 10 mg/kg cA2 group and the TNV148-treatedGroups were significantly higher at weeks 7 and 8, there were nosignificant differences in AI between the 1 mg/kg cA2, 1 mg/kg TNV148and 1 mg/kg TNV148B at any point in the study.

FIG. 18 shows an overview of the 9 stages of the golimumab manufacturingprocess.

FIG. 19 shows a flow diagram of Stage 1 manufacturing process for thepreculture and expansion steps, including the in-process controls andprocess monitoring tests.

FIG. 20 shows a flow diagram of Stage 2 manufacturing process steps,including the in-process controls and process monitoring tests.

FIG. 21 shows a representative HPLC chromatogram for golimumab expressedin Sp2/0 cells, using an HPLC method with fluorescence detection. Peaksassociated with different species are labeled. The * indicates a systempeak that is not associated with golimumab.

FIG. 22 shows a representative deconvoluted mass spectrum for IRMAanalysis of golimumab produced in Sp2/0 cells.

FIG. 23 shows a representative cIEF electropherogram profile ofgolimumab expressed in Sp2/0 cells, with the four major peaks labeled asC, 1, 2, and 3 and minor peak labeled B. Internal standards of pI 7.6and 9.5 are also labeled. A graphic representing the generalrelationship between cIEF peaks and decreasing negative charge/degree ofsialylation is also shown.

FIG. 24 shows a diagrammatic overview of some of the primary N-linkedoligosaccharide species in golimumab IgG. The role of some of theenzymes in the glycosylation maturation process and role of somedivalent cations (e.g. Mn²⁺ as a co-factor and Cu²⁺ as an inhibitor ofGalTI) are also shown (see, e.g., Biotechnol Bioeng. 2007 Feb. 15;96(3):538-49; Curr Drug Targets. 2008 April; 9(4):292-309; J Biochem MolBiol. 2002 May 31; 35(3):330-6). Note that species with terminal sialicacid (S1 and S2) are charged species and species lacking the terminalsialic acid (G0F, G1F, and G2F) are neutral species, but generation ofcharged species depends on the presence of the galactose in G1F and G2Fadded by the GalT1 enzyme.

FIG. 25 shows representative HPLC chromatograms for the oligosaccharideprofiles of golimumab expressed in Sp2/0 cells and golimumab expressedin CHO cells. Peaks of the eluting anthranilic acid-labeled N-glycansare identified with hash marks for all peaks above baseline. Bracketsand/or labels are used to indicate groups of peaks corresponding to theTotal Neutral, Total Charged, Monosialylated, and Disialylatedoligosaccharide species.

DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising anti-TNFantibodies having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 and manufacturing processes forproducing such anti-TNF antibodies.

As used herein, an “anti-tumor necrosis factor alpha antibody,”“anti-TNF antibody,” “anti-TNF antibody portion,” or “anti-TNF antibodyfragment” and/or “anti-TNF antibody variant” and the like include anyprotein or peptide containing molecule that comprises at least a portionof an immunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, or at least one portion of an TNFreceptor or binding protein, which can be incorporated into an antibodyof the present invention. Such antibody optionally further affects aspecific ligand, such as but not limited to where such antibodymodulates, decreases, increases, antagonizes, agonizes, mitigates,alleviates, blocks, inhibits, abrogates and/or interferes with at leastone TNF activity or binding, or with TNF receptor activity or binding,in vitro, in situ and/or in vivo. As a non-limiting example, a suitableanti-TNF antibody, specified portion or variant of the present inventioncan bind at least one TNF, or specified portions, variants or domainsthereof. A suitable anti-TNF antibody, specified portion, or variant canalso optionally affect at least one of TNF activity or function, such asbut not limited to, RNA, DNA or protein synthesis, TNF release, TNFreceptor signaling, membrane TNF cleavage, TNF activity, TNF productionand/or synthesis. The term “antibody” is further intended to encompassantibodies, digestion fragments, specified portions and variantsthereof, including antibody mimetics or comprising portions ofantibodies that mimic the structure and/or function of an antibody orspecified fragment or portion thereof, including single chain antibodiesand fragments thereof. Functional fragments include antigen-bindingfragments that bind to a mammalian TNF. For example, antibody fragmentscapable of binding to TNF or portions thereof, including, but notlimited to Fab (e.g., by papain digestion), Fab′ (e.g., by pepsindigestion and partial reduction) and F(ab′)₂ (e.g., by pepsindigestion), facb (e.g., by plasmin digestion), pFc′ (e.g., by pepsin orplasmin digestion), Fd (e.g., by pepsin digestion, partial reduction andreaggregation), Fv or scFv (e.g., by molecular biology techniques)fragments, are encompassed by the invention (see, e.g., Colligan,Immunology, supra).

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the CH₁ domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques or can be prepared as a contiguousprotein using genetic engineering techniques.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (e.g., CDR, framework, C_(L),C_(H) domains (e.g., C_(H)1, C_(H)2, and C_(H)3), hinge, (V_(L), V_(H)))is substantially non-immunogenic in humans, with only minor sequencechanges or variations. Similarly, antibodies designated primate (monkey,baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig,hamster, and the like) and other mammals designate such species,sub-genus, genus, sub-family, family specific antibodies. Further,chimeric antibodies include any combination of the above. Such changesor variations optionally and preferably retain or reduce theimmunogenicity in humans or other species relative to non-modifiedantibodies. Thus, a human antibody is distinct from a chimeric orhumanized antibody. It is pointed out that a human antibody can beproduced by a non-human animal or prokaryotic or eukaryotic cell that iscapable of expressing functionally rearranged human immunoglobulin(e.g., heavy chain and/or light chain) genes. Further, when a humanantibody is a single chain antibody, it can comprise a linker peptidethat is not found in native human antibodies. For example, an Fv cancomprise a linker peptide, such as two to about eight glycine or otheramino acid residues, which connects the variable region of the heavychain and the variable region of the light chain. Such linker peptidesare considered to be of human origin.

Bispecific (e.g., DuoBody®), heterospecific, heteroconjugate or similarantibodies can also be used that are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for at least one TNF protein, the other one is for anyother antigen. Methods for making bispecific antibodies are known in theart. Traditionally, the recombinant production of bispecific antibodiesis based on the co-expression of two immunoglobulin heavy chain-lightchain pairs, where the two heavy chains have different specificities(Milstein and Cuello, Nature 305:537 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of 10 different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule, which is usually done by affinitychromatography steps, can be cumbersome with low product yields anddifferent strategies have been developed to facilitate bispecificantibody production.

Full length bispecific antibodies can be generated for example using Fabarm exchange (or half molecule exchange) between two monospecificbivalent antibodies by introducing substitutions at the heavy chain CH3interface in each half molecule to favor heterodimer formation of twoantibody half molecules having distinct specificity either in vitro incell-free environment or using co-expression. The Fab arm exchangereaction is the result of a disulfide-bond isomerization reaction anddissociation-association of CH3 domains. The heavy-chain disulfide bondsin the hinge regions of the parent monospecific antibodies are reduced.The resulting free cysteines of one of the parent monospecificantibodies form an inter heavy-chain disulfide bond with cysteineresidues of a second parent monospecific antibody molecule andsimultaneously CH3 domains of the parent antibodies release and reformby dissociation-association. The CH3 domains of the Fab arms may beengineered to favor heterodimerization over homodimerization. Theresulting product is a bispecific antibody having two Fab arms or halfmolecules which each can bind a distinct epitope.

“Homodimerization” as used herein refers to an interaction of two heavychains having identical CH3 amino acid sequences. “Homodimer” as usedherein refers to an antibody having two heavy chains with identical CH3amino acid sequences.

“Heterodimerization” as used herein refers to an interaction of twoheavy chains having non-identical CH3 amino acid sequences.“Heterodimer” as used herein refers to an antibody having two heavychains with non-identical CH3 amino acid sequences.

The “knob-in-hole” strategy (see, e.g., PCT Intl. Publ. No. WO2006/028936) can be used to generate full length bispecific antibodies.Briefly, selected amino acids forming the interface of the CH3 domainsin human IgG can be mutated at positions affecting CH3 domaininteractions to promote heterodimer formation. An amino acid with asmall side chain (hole) is introduced into a heavy chain of an antibodyspecifically binding a first antigen and an amino acid with a large sidechain (knob) is introduced into a heavy chain of an antibodyspecifically binding a second antigen. After co-expression of the twoantibodies, a heterodimer is formed as a result of the preferentialinteraction of the heavy chain with a “hole” with the heavy chain with a“knob”. Exemplary CH3 substitution pairs forming a knob and a hole are(expressed as modified position in the first CH3 domain of the firstheavy chain/modified position in the second CH3 domain of the secondheavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T,T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.

Other strategies such as promoting heavy chain heterodimerization usingelectrostatic interactions by substituting positively charged residuesat one CH3 surface and negatively charged residues at a second CH3surface may be used, as described in US Pat. Publ. No. US2010/0015133;US Pat. Publ. No. US2009/0182127; US Pat. Publ. No. US2010/028637 or USPat. Publ. No. US2011/0123532. In other strategies, heterodimerizationmay be promoted by following substitutions (expressed as modifiedposition in the first CH3 domain of the first heavy chain/modifiedposition in the second CH3 domain of the second heavy chain):L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V,T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, orT350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in U.S.Pat. Publ. No. US2012/0149876 or U.S. Pat. Publ. No. US2013/0195849.

In addition to methods described above, bispecific antibodies can begenerated in vitro in a cell-free environment by introducingasymmetrical mutations in the CH3 regions of two monospecifichomodimeric antibodies and forming the bispecific heterodimeric antibodyfrom two parent monospecific homodimeric antibodies in reducingconditions to allow disulfide bond isomerization according to methodsdescribed in Intl. Pat. Publ. No. WO2011/131746. In the methods, thefirst monospecific bivalent antibody and the second monospecificbivalent antibody are engineered to have certain substitutions at theCH3 domain that promoter heterodimer stability; the antibodies areincubated together under reducing conditions sufficient to allow thecysteines in the hinge region to undergo disulfide bond isomerization;thereby generating the bispecific antibody by Fab arm exchange. Theincubation conditions may optimally be restored to non-reducing.Exemplary reducing agents that may be used are 2-mercaptoethylamine(2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione,tris(2-carboxyethyl)phosphine (TCEP), L-cysteine andbeta-mercaptoethanol, preferably a reducing agent selected from thegroup consisting of: 2-mercaptoethylamine, dithiothreitol andtris(2-carboxyethyl)phosphine. For example, incubation for at least 90min at a temperature of at least 20° C. in the presence of at least 25mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH offrom 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.

Anti-TNF antibodies (also termed TNF antibodies) useful in the methodsand compositions of the present invention can optionally becharacterized by high affinity binding to TNF and optionally andpreferably having low toxicity. In particular, an antibody, specifiedfragment or variant of the invention, where the individual components,such as the variable region, constant region and framework, individuallyand/or collectively, optionally and preferably possess lowimmunogenicity, is useful in the present invention. The antibodies thatcan be used in the invention are optionally characterized by theirability to treat patients for extended periods with measurablealleviation of symptoms and low and/or acceptable toxicity. Low oracceptable immunogenicity and/or high affinity, as well as othersuitable properties, can contribute to the therapeutic results achieved.“Low immunogenicity” is defined herein as raising significant HAHA, HACAor HAMA responses in less than about 75%, or preferably less than about50% of the patients treated and/or raising low titres in the patienttreated (less than about 300, preferably less than about 100 measuredwith a double antigen enzyme immunoassay) (Elliott et al., Lancet344:1125-1127 (1994), entirely incorporated herein by reference).

Utility:

The isolated nucleic acids of the present invention can be used forproduction of at least one anti-TNF antibody or specified variantthereof, which can be used to measure or effect in an cell, tissue,organ or animal (including mammals and humans), to diagnose, monitor,modulate, treat, alleviate, help prevent the incidence of, or reduce thesymptoms of, at least one TNF condition, selected from, but not limitedto, at least one of an immune disorder or disease, a cardiovasculardisorder or disease, an infectious, malignant, and/or neurologicdisorder or disease.

Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least oneanti-TNF antibody to a cell, tissue, organ, animal or patient in need ofsuch modulation, treatment, alleviation, prevention, or reduction insymptoms, effects or mechanisms. The effective amount can comprise anamount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple orcontinuous administration, or to achieve a serum concentration of0.01-5000 μg/ml serum concentration per single, multiple, or continuousadministration, or any effective range or value therein, as done anddetermined using known methods, as described herein or known in therelevant arts. Citations. All publications or patents cited herein areentirely incorporated herein by reference as they show the state of theart at the time of the present invention and/or to provide descriptionand enablement of the present invention. Publications refer to anyscientific or patent publications, or any other information available inany media format, including all recorded, electronic or printed formats.The following references are entirely incorporated herein by reference:Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley& Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning:A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989);Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor,N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology,John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., CurrentProtocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

Antibodies of the Present Invention:

At least one anti-TNF antibody of the present invention comprising allof the heavy chain variable CDR regions of SEQ ID NOS:1, 2 and 3 and/orall of the light chain variable CDR regions of SEQ ID NOS:4, 5 and 6 canbe optionally produced by a cell line, a mixed cell line, animmortalized cell or clonal population of immortalized cells, as wellknown in the art. See, e.g., Ausubel, et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001);Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor, N.Y. (1989); Harlow and Lane, antibodies, aLaboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al.,eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY(1994-2001); Colligan et al., Current Protocols in Protein Science, JohnWiley & Sons, NY, N.Y., (1997-2001), each entirely incorporated hereinby reference.

Human antibodies that are specific for human TNF proteins or fragmentsthereof can be raised against an appropriate immunogenic antigen, suchas isolated and/or TNF protein or a portion thereof (including syntheticmolecules, such as synthetic peptides). Other specific or generalmammalian antibodies can be similarly raised. Preparation of immunogenicantigens, and monoclonal antibody production can be performed using anysuitable technique.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or thelike, or heteromylomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart. See, e.g., www.atcc.org, www.lifetech.com., and the like, withantibody producing cells, such as, but not limited to, isolated orcloned spleen, peripheral blood, lymph, tonsil, or other immune or Bcell containing cells, or any other cells expressing heavy or lightchain constant or variable or framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference.

Antibody producing cells can also be obtained from the peripheral bloodor, preferably the spleen or lymph nodes, of humans or other suitableanimals that have been immunized with the antigen of interest. Any othersuitable host cell can also be used for expressing heterologous orendogenous nucleic acid encoding an antibody, specified fragment orvariant thereof, of the present invention. The fused cells (hybridomas)or recombinant cells can be isolated using selective culture conditionsor other suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods. Cells which produce antibodies with thedesired specificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK;BioInvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., EP 368,684, PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260 (May 12, 1994); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; (Scripps); EP 614 989(MorphoSys); WO95/16027 (BioInvent); WO88/06630; WO90/3809 (Dyax); U.S.Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323,5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803, EP590 689 (Ixsys, now Applied Molecular Evolution (AME), each entirelyincorporated herein by reference) or that rely upon immunization oftransgenic animals (e.g., SCID mice, Nguyen et al., Microbiol. Immunol.41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118(1996); Eren et al., Immunol. 93:154-161 (1998), each entirelyincorporated by reference as well as related patents and applications)that are capable of producing a repertoire of human antibodies, as knownin the art and/or as described herein. Such techniques, include, but arenot limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci.USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA,95:14130-14135 (November 1998)); single cell antibody producingtechnologies (e.g., selected lymphocyte antibody method (“SLAM”) (U.S.Pat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcooket al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gelmicrodroplet and flow cytometry (Powell et al., Biotechnol. 8:333-337(1990); One Cell Systems, Cambridge, Mass.; Gray et al., J. Imm. Meth.182:155-163 (1995); Kenny et al., Bio/Technol. 13:787-790 (1995));B-cell selection (Steenbakkers et al., Molec. Biol. Reports 19:125-134(1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization inHybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V.,Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies canalso be used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcewhich is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence.

Known human Ig sequences are disclosed in numerous publications andwebsites,

for example:

www.ncbi.nlm.nih.gov/entrez/query.fcgi;

www.atcc.org/phage/hdb.html;

www.sciquest.com/;

www.abcam.com/;

www.antibodyresource.com/onlinecomp.html;

www.publiciastate.edu/˜pedro/research tools.html;

www.mgen.uni-heidelberg.de/SD/IT/IT.html;

www.whfreeman.com/immunology/CH05/kuby05.htm;

www.library.thinkquest.org/12429/Immune/Antibody.html;

www.hhmi.org/grants/lectures/1996/vlab/;

www.path.cam.ac.uk/˜mrc7/mikeimages.html;

www.antibodyresource.com/;

www.mcb.harvard.edu/BioLinks/Immunology.html.

www.immunologylink.com/;

www.pathbox.wusthedu/˜hcenter/index.html;

www.biotech.ufl.edu/˜hcl/;

www.pebio.com/pa/340913/340913.html;

www.nal.usda.gov/awic/pubs/antibody/;

www.m.ehime-u.ac.jp/˜yasuhito/Elisa.html;

www.biodesign.com/table.asp;

www.icnet.uk/axp/facs/davies/links.html;

www.biotech.ufl.edu/˜fccl/protocol.html;

www.isac-net.org/sitesgeo.html;

www.aximtl.imt.uni-marburg.de/˜rek/AEPStart.html;

www.baserv.uci.kun.nl/˜jraats/linksl.html;

www.recab.uni-hd.de/immuno.bme.nwu.edu/;

www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;

www.ibt.unam.mx/virNmice.html; imgt.cnusc.fr:8104/;

www.biochem.ucl.ac.uk/martin/abs/index.html; antibody.bath.ac.uk/;

www.abgen.cvm.tamu.edu/lab/

www.abgen.html;

www.unizh ch/˜honegger/AHOseminar/Slide01.html;

www.cryst.bbk.ac.uk/˜ubcg07s/;

www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;

www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html;

www.ibt.unam.mx/viestructure/stataim.html;

www.biosci.missouri.edu/smithgp/index.html;

www.cryst.bioc.cam.ac.uk/˜fmolina/Web-pages/Pept/spottech.html;

www.jerini.de/frproducts.html;

www.patents.ibm.com/ibm.html.Kabat et al.,

Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. Generally, part or all of the non-human or human CDRsequences are maintained while the non-human sequences of the variableand constant regions are replaced with human or other amino acids.antibodies can also optionally be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding Humanization or engineering of antibodies ofthe present invention can be performed using any known method, such asbut not limited to those described in, Winter (Jones et al., Nature321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen etal., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296(1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al.,Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993), U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514,5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766886, 5714352, 6204023,6180370, 5693762, 5530101, 5585089, 5225539; 4816567, PCT/: US98/16280,US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134,GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, each entirelyincorporated herein by reference, included references cited therein.

The anti-TNF antibody can also be optionally generated by immunizationof a transgenic animal (e.g., mouse, rat, hamster, non-human primate,and the like) capable of producing a repertoire of human antibodies, asdescribed herein and/or as known in the art. Cells that produce a humananti-TNF antibody can be isolated from such animals and immortalizedusing suitable methods, such as the methods described herein.

Transgenic mice that can produce a repertoire of human antibodies thatbind to human antigens can be produced by known methods (e.g., but notlimited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126,5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al.;Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg etal. WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585,Kucherlapate et al. WO 96/34096, Kucherlapate et al. EP 0463 151 B1,Kucherlapate et al. EP 0710 719 A1, Surani et al. U.S. Pat. No.5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438 474B1, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440 A,Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int. Immunol.6(4)579-591 (1994), Green et al, Nature Genetics 7:13-21 (1994), Mendezet al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic AcidsResearch 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad SciUSA 90(8)3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1):65-93(1995) and Fishwald et al., Nat Biotechnol 14(7):845-851 (1996), whichare each entirely incorporated herein by reference). Generally, thesemice comprise at least one transgene comprising DNA from at least onehuman immunoglobulin locus that is functionally rearranged, or which canundergo functional rearrangement. The endogenous immunoglobulin loci insuch mice can be disrupted or deleted to eliminate the capacity of theanimal to produce antibodies encoded by endogenous genes.

Screening antibodies for specific binding to similar proteins orfragments can be conveniently achieved using peptide display libraries.This method involves the screening of large collections of peptides forindividual members having the desired function or structure. antibodyscreening of peptide display libraries is well known in the art. Thedisplayed peptide sequences can be from 3 to 5000 or more amino acids inlength, frequently from 5-100 amino acids long, and often from about 8to 25 amino acids long. In addition to direct chemical synthetic methodsfor generating peptide libraries, several recombinant DNA methods havebeen described. One type involves the display of a peptide sequence onthe surface of a bacteriophage or cell. Each bacteriophage or cellcontains the nucleotide sequence encoding the particular displayedpeptide sequence. Such methods are described in PCT Patent PublicationNos. 91/17271, 91/18980, 91/19818, and 93/08278. Other systems forgenerating libraries of peptides have aspects of both in vitro chemicalsynthesis and recombinant methods. See, PCT Patent Publication Nos.92/05258, 92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754;and 5,643,768. Peptide display libraries, vector, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.), and Cambridge antibody Technologies (Cambridgeshire, UK). See,e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203,5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260,5,856,456, assigned to Enzon; U.S. Pat. Nos. 5,223,409, 5,403,484,5,571,698, 5,837,500, assigned to Dyax, 5427908, 5580717, assigned toAffymax; 5885793, assigned to Cambridge antibody Technologies; 5750373,assigned to Genentech, 5618920, 5595898, 5576195, 5698435, 5693493,5698417, assigned to Xoma, Colligan, supra; Ausubel, supra; or Sambrook,supra, each of the above patents and publications entirely incorporatedherein by reference.

Antibodies of the present invention can also be prepared using at leastone anti-TNF antibody encoding nucleic acid to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. Such animals can be providedusing known methods. See, e.g., but not limited to, U.S. Pat. Nos.5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362;5,304,489, and the like, each of which is entirely incorporated hereinby reference.

Antibodies of the present invention can additionally be prepared usingat least one anti-TNF antibody encoding nucleic acid to providetransgenic plants and cultured plant cells (e.g., but not limited totobacco and maize) that produce such antibodies, specified portions orvariants in the plant parts or in cells cultured therefrom. As anon-limiting example, transgenic tobacco leaves expressing recombinantproteins have been successfully used to provide large amounts ofrecombinant proteins, e.g., using an inducible promoter. See, e.g.,Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencecited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to know methods. See also,e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October,1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., PlantPhysiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans.22:940-944 (1994); and references cited therein. See, also generally forplant expression of antibodies, but not limited to, Each of the abovereferences is entirely incorporated herein by reference.

The antibodies of the invention can bind human TNF with a wide range ofaffinities (K_(D)). In a preferred embodiment, at least one human mAb ofthe present invention can optionally bind human TNF with high affinity.For example, a human mAb can bind human TNF with a K_(D) equal to orless than about 10⁻⁷ M, such as but not limited to, 0.1-9.9 (or anyrange or value therein)×10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ orany range or value therein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

Nucleic Acid Molecules.

Using the information provided herein, such as the nucleotide sequencesencoding at least 70-100% of the contiguous amino acids of at least oneof SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, specified fragments, variants orconsensus sequences thereof, or a deposited vector comprising at leastone of these sequences, a nucleic acid molecule of the present inventionencoding at least one anti-TNF antibody comprising all of the heavychain variable CDR regions of SEQ ID NOS:1, 2 and 3 and/or all of thelight chain variable CDR regions of SEQ ID NOS:4, 5 and 6 can beobtained using methods described herein or as known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain (e.g., SEQ ID NOS:1-3) or light chain (e.g.,SEQ ID NOS: 4-6); nucleic acid molecules comprising the coding sequencefor an anti-TNF antibody or variable region (e.g., SEQ ID NOS:7,8); andnucleic acid molecules which comprise a nucleotide sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode at least one anti-TNFantibody as described herein and/or as known in the art. Of course, thegenetic code is well known in the art. Thus, it would be routine for oneskilled in the art to generate such degenerate nucleic acid variantsthat code for specific anti-TNF antibodies of the present invention.See, e.g., Ausubel, et al., supra, and such nucleic acid variants areincluded in the present invention. Non-limiting examples of isolatednucleic acid molecules of the present invention include SEQ ID NOS:10,11, 12, 13, 14, 15, corresponding to non-limiting examples of a nucleicacid encoding, respectively, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LCCDR2, LC CDR3, HC variable region and LC variable region.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-TNF antibody can include,but are not limited to, those encoding the amino acid sequence of anantibody fragment, by itself; the coding sequence for the entireantibody or a portion thereof; the coding sequence for an antibody,fragment or portion, as well as additional sequences, such as the codingsequence of at least one signal leader or fusion peptide, with orwithout the aforementioned additional coding sequences, such as at leastone intron, together with additional, non-coding sequences, includingbut not limited to, non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences that play a role in transcription,mRNA processing, including splicing and polyadenylation signals (forexample—ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding anantibody can be fused to a marker sequence, such as a sequence encodinga peptide that facilitates purification of the fused antibody comprisingan antibody fragment or portion.

Polynucleotides which Selectively Hybridize to a Polynucleotide asDescribed Herein.

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides of the present inventioncan be used to identify, isolate, or amplify partial or full-lengthclones in a deposited library. In some embodiments, the polynucleotidesare genomic or cDNA sequences isolated, or otherwise complementary to, acDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably at least 85% or 90% full-length sequences, andmore preferably at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of an antibody encoded by the polynucleotides described herein.The polynucleotides of this invention embrace nucleic acid sequencesthat can be employed for selective hybridization to a polynucleotideencoding an antibody of the present invention. See, e.g., Ausubel,supra; Colligan, supra, each entirely incorporated herein by reference.

Construction of Nucleic Acids.

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra).

Recombinant Methods for Constructing Nucleic Acids.

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra).

Nucleic Acid Screening and Isolation Methods.

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; U.S. Pat.No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson, et al.; U.S.Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 to Gyllensten, etal; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No. 4,994,370to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S. Pat. No.4,656,134 to Ringold) and RNA mediated amplification that usesanti-sense RNA to the target sequence as a template for double-strandedDNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with the tradename NASBA), the entire contents of which references are incorporatedherein by reference. (See, e.g., Ausubel, supra; or Sambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can beused to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids.

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences.

Recombinant Expression Cassettes.

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example a cDNA or a genomicsequence encoding an antibody of the present invention, can be used toconstruct a recombinant expression cassette that can be introduced intoat least one desired host cell. A recombinant expression cassette willtypically comprise a polynucleotide of the present invention operablylinked to transcriptional initiation regulatory sequences that willdirect the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in intron) of a non-heterologous formof a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

Vectors and Host Cells.

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one anti-TNF antibody by recombinant techniques, as is wellknown in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al.,supra, each entirely incorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating site at the beginning and a termination codon(e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNAto be translated, with UAA and UAG preferred for mammalian or eukaryoticcell expression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe affected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one antibody of the present invention can be expressed in amodified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of an antibody to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage. Also, peptide moieties can beadded to an antibody of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofan antibody or at least one fragment thereof. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Chapters17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention.

Alternatively, nucleic acids of the present invention can be expressedin a host cell by turning on (by manipulation) in a host cell thatcontains endogenous DNA encoding an antibody of the present invention.Such methods are well known in the art, e.g., as described in U.S. Pat.Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirelyincorporated herein by reference.

Cells useful for the production of the antibodies, specified portions orvariants thereof, include mammalian cells. Mammalian cell systems oftenwill be cultured in the form of monolayers of cells, but the cells canalso be adapted to grow in suspension, e.g., in shake flasks orbioreactors. A number of suitable host cell lines capable of expressingintact glycosylated proteins have been developed in the art, and includethe COS-1 (e.g., ATCC® CRL-1650), COS-7 (e.g., ATCC® CRL-1651), HEK293,BHK21 (e.g., ATCC® CCL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g.,ATCC CRL-26) cell lines, Chinese Hamster Ovary (CHO), Hep G2,P3X63Ag8.653, Sp2/0-Ag14, HeLa cells and the like, which are readilyavailable from, for example, American Type Culture Collection, Manassas,Va. In certain embodiments host cells include CHO cells and cells oflymphoid origin such as myeloma and lymphoma cells, e.g., CHO-K1 cells,P3X63Ag8.653 cells (ATCC® CRL-1580), and Sp2/0-Ag14 cells (ATCC®CRL-1581).

CHO Cell Lines

Despite the availability of several other mammalian cell lines, amajority of recombinant therapeutic proteins produced today are made inChinese hamster ovary (CHO) cells (Jayapal K P, et al. Recombinantprotein therapeutics from CHO cells-20 years and counting. Chem EngProg. 2007; 103:40-47; Kunert R, Reinhart D. Advances in recombinantantibody manufacturing. Appl Microbiol Biotechnol. 2016;100(8):3451-61). Their strengths include, e.g., robust growth asadherent cells or in suspension, adaptability to serum-free andchemically defined media, high productivity, and an established historyof regulatory approval for therapeutic recombinant protein production.They are also very amenable to genetic modifications and the methods forcell transfection, recombinant protein expression, and clone selectionare all well characterized. CHO cells can also provide human-compatiblepost-translational modifications. As used herein, “CHO cells” include,but are not limited to, e.g., CHO-DG44, CHO-K1, CHO-M, CHO-S, CHO GSknockout, and modifications and derivatives thereof.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomasor other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Purification of an Antibody.

An anti-TNF antibody can be recovered and purified from recombinant cellcultures by well-known methods including, but not limited to, protein Apurification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

Anti-TNF Antibodies

The isolated antibodies of the present invention, comprising all of theheavy chain variable CDR regions of SEQ ID NOS:1, 2 and 3 and/or all ofthe light chain variable CDR regions of SEQ ID NOS:4, 5 and 6, compriseantibody amino acid sequences disclosed herein encoded by any suitablepolynucleotide, or any isolated or prepared antibody. Preferably, thehuman antibody or antigen-binding fragment binds human TNF and, therebypartially or substantially neutralizes at least one biological activityof the protein. An antibody, or specified portion or variant thereof,that partially or preferably substantially neutralizes at least onebiological activity of at least one TNF protein or fragment can bind theprotein or fragment and thereby inhibit activities mediated through thebinding of TNF to the TNF receptor or through other TNF-dependent ormediated mechanisms. As used herein, the term “neutralizing antibody”refers to an antibody that can inhibit an TNF-dependent activity byabout 20-120%, preferably by at least about 10, 20, 30, 40, 50, 55, 60,65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or moredepending on the assay. The capacity of an anti-TNF antibody to inhibitan TNF-dependent activity is preferably assessed by at least onesuitable TNF protein or receptor assay, as described herein and/or asknown in the art. A human antibody of the invention can be of any class(IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa orlambda light chain. In one embodiment, the human antibody comprises anIgG heavy chain or defined fragment, for example, at least one ofisotypes, IgG1, IgG2, IgG3 or IgG4. Antibodies of this type can beprepared by employing a transgenic mouse or other transgenic non-humanmammal comprising at least one human light chain (e.g., IgG, IgA) andIgM (e.g., γ1, γ2, γ3, γ4) transgenes as described herein and/or asknown in the art. In another embodiment, the anti-human TNF humanantibody comprises an IgG1 heavy chain and a IgG1 light chain.

As used herein, the terms “antibody” or “antibodies”, include biosimilarantibody molecules approved under the Biologics Price Competition andInnovation Act of 2009 (BPCI Act) and similar laws and regulationsglobally. Under the BPCI Act, an antibody may be demonstrated to bebiosimilar if data show that it is “highly similar” to the referenceproduct notwithstanding minor differences in clinically inactivecomponents and are “expected” to produce the same clinical result as thereference product in terms of safety, purity and potency (EndocrinePractice: February 2018, Vol. 24, No. 2, pp. 195-204). These biosimilarantibody molecules are provided an abbreviated approval pathway, wherebythe applicant relies upon the innovator reference product's clinicaldata to secure regulatory approval. Compared to the original innovatorreference antibody that was FDA approved based on successful clinicaltrials, a biosimilar antibody molecule is referred to herein as a“follow-on biologic”. As presented herein, SIMPONI® (golimumab) is theoriginal innovator reference anti-TNF antibody that was FDA approvedbased on successful clinical trials. Golimumab has been on sale in theUnited States since 2009.

At least one antibody of the invention binds at least one specifiedepitope specific to at least one TNF protein, subunit, fragment, portionor any combination thereof. The at least one epitope can comprise atleast one antibody binding region that comprises at least one portion ofsaid protein, which epitope is preferably comprised of at least oneextracellular, soluble, hydrophilic, external or cytoplasmic portion ofsaid protein. The at least one specified epitope can comprise anycombination of at least one amino acid sequence of at least 1-3 aminoacids to the entire specified portion of contiguous amino acids of theSEQ ID NO:9.

Generally, the human antibody or antigen-binding fragment of the presentinvention will comprise an antigen-binding region that comprises atleast one human complementarity determining region (CDR1, CDR2 and CDR3)or variant of at least one heavy chain variable region and at least onehuman complementarity determining region (CDR1, CDR2 and CDR3) orvariant of at least one light chain variable region. As a non-limitingexample, the antibody or antigen-binding portion or variant can compriseat least one of the heavy chain CDR3 having the amino acid sequence ofSEQ ID NO:3, and/or a light chain CDR3 having the amino acid sequence ofSEQ ID NO:6. In a particular embodiment, the antibody or antigen-bindingfragment can have an antigen-binding region that comprises at least aportion of at least one heavy chain CDR (i.e., CDR1, CDR2 and/or CDR3)having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3(e.g., SEQ ID NOS:1, 2, and/or 3). In another particular embodiment, theantibody or antigen-binding portion or variant can have anantigen-binding region that comprises at least a portion of at least onelight chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acidsequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 4,5, and/or 6). In a preferred embodiment the three heavy chain CDRs andthe three light chain CDRs of the antibody or antigen-binding fragmenthave the amino acid sequence of the corresponding CDR of at least one ofmAb TNV148, TNV14, TNV15, TNV196, TNV118, TNV32, TNV86, as describedherein. Such antibodies can be prepared by chemically joining togetherthe various portions (e.g., CDRs, framework) of the antibody usingconventional techniques, by preparing and expressing a (i.e., one ormore) nucleic acid molecule that encodes the antibody using conventionaltechniques of recombinant DNA technology or by using any other suitablemethod.

The anti-TNF antibody can comprise at least one of a heavy or lightchain variable region having a defined amino acid sequence. For example,in a preferred embodiment, the anti-TNF antibody comprises at least oneof heavy chain variable region, optionally having the amino acidsequence of SEQ ID NO:7 and/or at least one light chain variable region,optionally having the amino acid sequence of SEQ ID NO:8. antibodiesthat bind to human TNF and that comprise a defined heavy or light chainvariable region can be prepared using suitable methods, such as phagedisplay (Katsube, Y., et al., Int J Mol. Med, 1(5):863-868 (1998)) ormethods that employ transgenic animals, as known in the art and/or asdescribed herein. For example, a transgenic mouse, comprising afunctionally rearranged human immunoglobulin heavy chain transgene and atransgene comprising DNA from a human immunoglobulin light chain locusthat can undergo functional rearrangement, can be immunized with humanTNF or a fragment thereof to elicit the production of antibodies. Ifdesired, the antibody producing cells can be isolated and hybridomas orother immortalized antibody-producing cells can be prepared as describedherein and/or as known in the art. Alternatively, the antibody,specified portion or variant can be expressed using the encoding nucleicacid or portion thereof in a suitable host cell.

The invention also relates to antibodies, antigen-binding fragments,immunoglobulin chains and CDRs comprising amino acids in a sequence thatis substantially the same as an amino acid sequence described herein.Preferably, such antibodies or antigen-binding fragments and antibodiescomprising such chains or CDRs can bind human TNF with high affinity(e.g., K_(D) less than or equal to about 10⁻⁹ M). Amino acid sequencesthat are substantially the same as the sequences described hereininclude sequences comprising conservative amino acid substitutions, aswell as amino acid deletions and/or insertions. A conservative aminoacid substitution refers to the replacement of a first amino acid by asecond amino acid that has chemical and/or physical properties (e.g.,charge, structure, polarity, hydrophobicity/hydrophilicity) that aresimilar to those of the first amino acid. Conservative substitutionsinclude replacement of one amino acid by another within the followinggroups: lysine (K), arginine (R) and histidine (H); aspartate (D) andglutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T),tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L),isoleucine (I), proline (P), phenylalanine (F), tryptophan (W),methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.

Amino Acid Codes.

The amino acids that make up anti-TNF antibodies of the presentinvention are often abbreviated. The amino acid designations can beindicated by designating the amino acid by its single letter code, itsthree letter code, name, or three nucleotide codon(s) as is wellunderstood in the art (see Alberts, B., et al., Molecular Biology of TheCell, Third Ed., Garland Publishing, Inc., New York, 1994):

SINGLE THREE THREE LETTER LETTER NUCLEOTIDE CODE CODE NAME CODON(S) AAla Alanine GCA, GCC, GCG, GCU C Cys Cysteine UGC, UGU D Asp Asparticacid GAC, GAU E Glu Glutamic acid GAA, GAG F Phe Phenylanine UUC, UUU GGly Glycine GGA, GGC, GGG, GGU H His Histidine CAC, CAU I Ile IsoleucineAUA, AUC, AUU K Lys Lysine AAA, AAG L Leu Leucine UUA, UUG, CUA, CUC,CUG, CUU M Met Methionine AUG N Asn Asparagine AAC, AAU P Pro ProlineCCA, CCC, CCG, CCU Q Gln Glutamine CAA, CAG R Arg Arginine AGA, AGG,CGA, CGC, CGG, CGU S Ser Serine AGC, AGU, UCA, UCC, UCG, UCU T ThrThreonine ACA, ACC, ACG, ACU V Val Valine GUA, GUC, GUG, GUU W TrpTryptophan UGG Y Tyr Tyrosine UAC, UAU

An anti-TNF antibody of the present invention can include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation, as specified herein.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for any given anti-TNF antibody, fragment or variant willnot be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, asspecified herein.

Amino acids in an anti-TNF antibody of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g.,Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science244:1081-1085 (1989)). The latter procedure introduces single alaninemutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least one TNF neutralizing activity. Sites that arecritical for antibody binding can also be identified by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992)and de Vos, et al., Science 255:306-312 (1992)).

Anti-TNF antibodies of the present invention can include, but are notlimited to, at least one portion, sequence or combination selected from1 to all of the contiguous amino acids of at least one of SEQ ID NOS:1,2, 3, 4, 5, 6.

A(n) anti-TNF antibody can further optionally comprise a polypeptide ofat least one of 70-100% of the contiguous amino acids of at least one ofSEQ ID NOS:7, 8.

In one embodiment, the amino acid sequence of an immunoglobulin chain,or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of thecorresponding chain of at least one of SEQ ID NOS:7, 8. For example, theamino acid sequence of a light chain variable region can be comparedwith the sequence of SEQ ID NO:8, or the amino acid sequence of a heavychain CDR3 can be compared with SEQ ID NO:7. Preferably, 70-100% aminoacid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or anyrange or value therein) is determined using a suitable computeralgorithm, as known in the art.

Exemplary heavy chain and light chain variable regions sequences areprovided in SEQ ID NOS: 7, 8. The antibodies of the present invention,or specified variants thereof, can comprise any number of contiguousamino acid residues from an antibody of the present invention, whereinthat number is selected from the group of integers consisting of from10-100% of the number of contiguous residues in an anti-TNF antibody.Optionally, this subsequence of contiguous amino acids is at least about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids inlength, or any range or value therein. Further, the number of suchsubsequences can be any integer selected from the group consisting offrom 1 to 20, such as at least 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-1000% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

The modified antibodies and antigen-binding fragments of the inventioncan comprise one or more organic moieties that are covalently bonded,directly or indirectly, to the antibody. Each organic moiety that isbonded to an antibody or antigen-binding fragment of the invention canindependently be a hydrophilic polymeric group, a fatty acid group or afatty acid ester group. As used herein, the term “fatty acid”encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, anantibody modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying antibodies of the invention can be linear or branched andinclude, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example, PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodies ofthe invention include, for example, n-dodecanoate (C₁₂, laurate),n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈, stearate),n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂, behenate),n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀), cis-Δ9-octadecanoate(C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate (C₂₀, arachidonate),octanedioic acid, tetradecanedioic acid, octadecanedioic acid,docosanedioic acid, and the like. Suitable fatty acid esters includemono-esters of dicarboxylic acids that comprise a linear or branchedlower alkyl group. The lower alkyl group can comprise from one to abouttwelve, preferably one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis(Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996);Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and themethods described in Hermanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

Anti-Idiotype Antibodies to Anti-Tnf Antibody Compositions.

In addition to monoclonal or chimeric anti-TNF antibodies, the presentinvention is also directed to an anti-idiotypic (anti-Id) antibodyspecific for such antibodies of the invention. An anti-Id antibody is anantibody which recognizes unique determinants generally associated withthe antigen-binding region of another antibody. The anti-Id can beprepared by immunizing an animal of the same species and genetic type(e.g. mouse strain) as the source of the Id antibody with the antibodyor a CDR containing region thereof. The immunized animal will recognizeand respond to the idiotypic determinants of the immunizing antibody andproduce an anti-Id antibody. The anti-Id antibody may also be used as an“immunogen” to induce an immune response in yet another animal,producing a so-called anti-anti-Id antibody.

Anti-Tnf Antibody Compositions.

The present invention also provides at least one anti-TNF antibodycomposition comprising at least one, at least two, at least three, atleast four, at least five, at least six or more anti-TNF antibodiesthereof, as described herein and/or as known in the art that areprovided in a non-naturally occurring composition, mixture or form. Suchcompositions comprise non-naturally occurring compositions comprising atleast one or two full length, C- and/or N-terminally deleted variants,domains, fragments, or specified variants, of the anti-TNF antibodyamino acid sequence selected from the group consisting of 70-100% of thecontiguous amino acids of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, orspecified fragments, domains or variants thereof. Preferred anti-TNFantibody compositions include at least one or two full length,fragments, domains or variants as at least one CDR or LBR containingportions of the anti-TNF antibody sequence of 70-100% of SEQ ID NOS:1,2, 3, 4, 5, 6, or specified fragments, domains or variants thereof.Further preferred compositions comprise 40-99% of at least one of70-100% of SEQ ID NOS:1, 2, 3, 4, 5, 6, or specified fragments, domainsor variants thereof. Such composition percentages are by weight, volume,concentration, molarity, or molality as liquid or dry solutions,mixtures, suspension, emulsions or colloids, as known in the art or asdescribed herein.

Anti-TNF antibody compositions of the present invention can furthercomprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising at least oneanti-TNF antibody to a cell, tissue, organ, animal or patient in need ofsuch modulation, treatment or therapy, optionally further comprising atleast one selected from at least one TNF antagonist (e.g., but notlimited to a TNF antibody or fragment, a soluble TNF receptor orfragment, fusion proteins thereof, or a small molecule TNF antagonist),an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose,azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquinesulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, anantiviral, a carbapenem, cephalosporin, a fluoroquinolone, a macrolide,a penicillin, a sulfonamide, a tetracycline, another antimicrobial), anantipsoriatic, a corticosteriod, an anabolic steroid, a diabetes relatedagent, a mineral, a nutritional, a thyroid agent, a vitamin, a calciumrelated hormone, an antidiarrheal, an antitussive, an antiemetic, anantiulcer, a laxative, an anticoagulant, an erythropoietin (e.g.,epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim(GM-CSF, Leukine), an immunization, an immunoglobulin, animmunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), agrowth hormone, a hormone replacement drug, an estrogen receptormodulator, a mydriatic, a cycloplegic, an alkylating agent, anantimetabolite, a mitotic inhibitor, a radiopharmaceutical, anantidepressant, antimanic agent, an antipsychotic, an anxiolytic, ahypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthmamedication, a beta agonist, an inhaled steroid, a leukotriene inhibitor,a methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha(Pulmozyme), a cytokine or a cytokine antagonist. Non-limiting examplesof such cytokines include, but are not limited to, any of IL-1 to IL-23.Suitable dosages are well known in the art. See, e.g., Wells et al.,eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange,Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000),each of which references are entirely incorporated herein by reference.

Such anti-cancer or anti-infectives can also include toxin moleculesthat are associated, bound, co-formulated or co-administered with atleast one antibody of the present invention. The toxin can optionallyact to selectively kill the pathologic cell or tissue. The pathologiccell can be a cancer or other cell. Such toxins can be, but are notlimited to, purified or recombinant toxin or toxin fragment comprisingat least one functional cytotoxic domain of toxin, e.g., selected fromat least one of ricin, diphtheria toxin, a venom toxin, or a bacterialtoxin. The term toxin also includes both endotoxins and exotoxinsproduced by any naturally occurring, mutant or recombinant bacteria orviruses which may cause any pathological condition in humans and othermammals, including toxin shock, which can result in death. Such toxinsmay include, but are not limited to, enterotoxigenic E. coli heat-labileenterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin,Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-1),Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcalenterotoxins and the like. Such bacteria include, but are not limitedto, strains of a species of enterotoxigenic E. coli (ETEC),enterohemorrhagic E. coli (e.g., strains of serotype O157:H7),Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcuspyogenes), Shigella species (e.g., Shigella dysenteriae, Shigellaflexneri, Shigella boydii, and Shigella sonnei), Salmonella species(e.g., Salmonella typhi, Salmonella cholera-suis, Salmonellaenteritidis), Clostridium species (e.g., Clostridium perfringens,Clostridium dificile, Clostridium botulinum), Campylobacter species(e.g., Campylobacter jejuni, Campylobacter fetus), Helicobacter species,(e.g., Helicobacter pylori), Aeromonas species (e.g., Aeromonas sobria,Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides,Yersinia enterocolitica, Vibrio species (e.g., Vibrio cholerae, Vibrioparahemolyticus), Klebsiella species, Pseudomonas aeruginosa, andStreptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds.,Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp239-254, Plenum Medical Book Co., New York (1991); Mandell et al,Principles and Practice of Infectious Diseases, 3d. Ed., ChurchillLivingstone, New York (1990); Berkow et al, eds., The Merck Manual, 16thedition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack et al, Science,248:705-711 (1990), the contents of which references are incorporatedentirely herein by reference.

Anti-TNF antibody compounds, compositions or combinations of the presentinvention can further comprise at least one of any suitable auxiliary,such as, but not limited to, diluent, binder, stabilizer, buffers,salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the anti-TNF antibody, fragment or variantcomposition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Anti-TNF antibody compositions can also include a buffer or a pHadjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid saltssuch as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts such as citrate.

Additionally, anti-TNF antibody compositions of the invention caninclude polymeric excipients/additives such as polyvinylpyrrolidones,ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the anti-TNF antibody, portion or variantcompositions according to the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed.,Williams & Williams, (1995), and in the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), the disclosuresof which are entirely incorporated herein by reference. Preferredcarrier or excipient materials are carbohydrates (e.g., saccharides andalditols) and buffers (e.g., citrate) or polymeric agents.

Formulations.

As noted above, the invention provides for stable formulations, which ispreferably a phosphate buffer with saline or a chosen salt, as well aspreserved solutions and formulations containing a preservative as wellas multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one anti-TNF antibody in apharmaceutically acceptable formulation. Preserved formulations containat least one known preservative or optionally selected from the groupconsisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concentration ormixture can be used as known in the art, such as 0.001-5%, or any rangeor value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1-2%m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol(e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g.,0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9,1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5,0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one anti-TNF antibody with the prescribed buffersand/or preservatives, optionally in an aqueous diluent, wherein saidpackaging material comprises a label that indicates that such solutioncan be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30,36, 40, 48, 54, 60, 66, 72 hours or greater. The invention furthercomprises an article of manufacture, comprising packaging material, afirst vial comprising lyophilized at least one anti-TNF antibody, and asecond vial comprising an aqueous diluent of prescribed buffer orpreservative, wherein said packaging material comprises a label thatinstructs a patient to reconstitute the at least one anti-TNF antibodyin the aqueous diluent to form a solution that can be held over a periodof twenty-four hours or greater.

The at least one anti-TNF antibody used in accordance with the presentinvention can be produced by recombinant means, including from mammaliancell or transgenic preparations, or can be purified from otherbiological sources, as described herein or as known in the art.

The range of at least one anti-TNF antibody in the product of thepresent invention includes amounts yielding upon reconstitution, if in awet/dry system, concentrations from about 1.0 μg/ml to about 1000 mg/ml,although lower and higher concentrations are operable and are dependenton the intended delivery vehicle, e.g., solution formulations willdiffer from transdermal patch, pulmonary, transmucosal, or osmotic ormicro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyols, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one anti-TNF antibody and a preservativeselected from the group consisting of phenol, m-cresol, p-cresol,o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one anti-TNF antibody andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one anti-TNF antibody inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized at least one anti-TNFantibody that is reconstituted with a second vial containing water, apreservative and/or excipients, preferably a phosphate buffer and/orsaline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2 to about 40° C. and retain the biologically activity of the proteinfor extended periods of time, thus, allowing a package label indicatingthat the solution can be held and/or used over a period of 6, 12, 18,24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used,such label can include use up to 1-12 months, one-half, one and a half,and/or two years.

The solutions of at least one anti-TNF antibody in the invention can beprepared by a process that comprises mixing at least one antibody in anaqueous diluent. Mixing is carried out using conventional dissolutionand mixing procedures. To prepare a suitable diluent, for example, ameasured amount of at least one antibody in water or buffer is combinedin quantities sufficient to provide the protein and optionally apreservative or buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one anti-TNFantibody that is reconstituted with a second vial containing the aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneanti-TNF antibody that is reconstituted with a second vial containingthe aqueous diluent. The clear solution in this case can be up to oneliter or even larger in size, providing a large reservoir from whichsmaller portions of the at least one antibody solution can be retrievedone or multiple times for transfer into smaller vials and provided bythe pharmacy or clinic to their customers and/or patients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution such as BD Pens, BDAutojector®, Humaject® NovoPen®, B-D® Pen, AutoPen®, and OptiPen®,GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen, Roferon Pen®,Biojector, iject, J-tip Needle-Free Injector®, Intraject Medi-Ject,e.g., as made or developed by:

Becton Dickensen (Franklin Lakes, N.J., www.bectondickenson.com),

Disetronic (Burgdorf, Switzerland, www.disetronic.com;

Bioject, Portland, Oreg. (www.bioject.com);

Weston Medical (Peterborough, UK, www.weston-medical.com),

Medi-Ject Corp (Minneapolis, Minn., www.mediject.com).

Recognized devices comprising a dual vial system include thosepen-injector systems for reconstituting a lyophilized drug in acartridge for delivery of the reconstituted solution such as theHumatroPen®.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute the at least one anti-TNF antibody in theaqueous diluent to form a solution and to use the solution over a periodof 2-24 hours or greater for the two vial, wet/dry, product. For thesingle vial, solution product, the label indicates that such solutioncan be used over a period of 2-24 hours or greater. The presentlyclaimed products are useful for human pharmaceutical product use.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one anti-TNF antibody and a selectedbuffer, preferably a phosphate buffer containing saline or a chosensalt. Mixing the at least one antibody and buffer in an aqueous diluentis carried out using conventional dissolution and mixing procedures. Toprepare a suitable formulation, for example, a measured amount of atleast one antibody in water or buffer is combined with the desiredbuffering agent in water in quantities sufficient to provide the proteinand buffer at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilized atleast one anti-TNF antibody that is reconstituted with a second vialcontaining a preservative or buffer and excipients in an aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

At least one anti-TNF antibody in either the stable or preservedformulations or solutions described herein, can be administered to apatient in accordance with the present invention via a variety ofdelivery methods including SC or IM injection; transdermal, pulmonary,transmucosal, implant, osmotic pump, cartridge, micro pump, or othermeans appreciated by the skilled artisan, as well-known in the art.

Therapeutic Applications.

The present invention also provides a method for modulating or treatingat least one TNF related disease, in a cell, tissue, organ, animal, orpatient, as known in the art or as described herein, using at least onedual integrin antibody of the present invention.

The present invention also provides a method for modulating or treatingat least one TNF related disease, in a cell, tissue, organ, animal, orpatient including, but not limited to, at least one of obesity, animmune related disease, a cardiovascular disease, an infectious disease,a malignant disease or a neurologic disease.

The present invention also provides a method for modulating or treatingat least one immune related disease, in a cell, tissue, organ, animal,or patient including, but not limited to, at least one of rheumatoidarthritis, juvenile, systemic onset juvenile rheumatoid arthritis,Ankylosing Spondylitis, ankylosing spondilitis, gastric ulcer,seronegative arthropathies, osteoarthritis, inflammatory bowel disease,ulcerative colitis, systemic lupus erythematosis, antiphospholipidsyndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonaryfibrosis, systemic vasculitis/wegener's granulomatosis, sarcoidosis,orchitis/vasectomy reversal procedures, allergic/atopic diseases,asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergicconjunctivitis, hypersensitivity pneumonitis, transplants, organtransplant rejection, graft-versus-host disease, systemic inflammatoryresponse syndrome, sepsis syndrome, gram positive sepsis, gram negativesepsis, culture negative sepsis, fungal sepsis, neutropenic fever,urosepsis, meningococcemia, trauma/hemorrhage, burns, ionizing radiationexposure, acute pancreatitis, adult respiratory distress syndrome,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitivity reactions, allergic rhinitis, hayfever, perennial rhinitis, conjunctivitis, endometriosis, asthma,urticaria, systemic anaphylaxis, dermatitis, pernicious anemia,hemolytic disease, thrombocytopenia, graft rejection of any organ ortissue, kidney transplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, primary biliary cirrhosis, thyroiditis,encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lungdisease, chronic obstructive pulmonary disease (COPD), familialhematophagocytic lymphohistiocytosis, dermatologic conditions,psoriasis, alopecia, nephrotic syndrome, nephritis, glomerularnephritis, acute renal failure, hemodialysis, uremia, toxicity,preeclampsia, okt3 therapy, anti-cd3 therapy, cytokine therapy,chemotherapy, radiation therapy (e.g., including but not limited toasthenia, anemia, cachexia, and the like), chronic salicylateintoxication, and the like. See, e.g., the Merck Manual, 12th-17thEditions, Merck & Company, Rahway, N.J. (1972, 1977, 1982, 1987, 1992,1999), Pharmacotherapy Handbook, Wells et al., eds., Second Edition,Appleton and Lange, Stamford, Conn. (1998, 2000), each entirelyincorporated by reference.

The present invention also provides a method for modulating or treatingat least one cardiovascular disease in a cell, tissue, organ, animal, orpatient, including, but not limited to, at least one of cardiac stunsyndrome, myocardial infarction, congestive heart failure, stroke,ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis,restenosis, diabetic arteriosclerotic disease, hypertension, arterialhypertension, renovascular hypertension, syncope, shock, syphilis of thecardiovascular system, heart failure, cor pulmonale, primary pulmonaryhypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter,atrial fibrillation (sustained or paroxysmal), post perfusion syndrome,cardiopulmonary bypass inflammation response, chaotic or multifocalatrial tachycardia, regular narrow QRS tachycardia, specificarrhythmias, ventricular fibrillation, His bundle arrhythmias,atrioventricular block, bundle branch block, myocardial ischemicdisorders, coronary artery disease, angina pectoris, myocardialinfarction, cardiomyopathy, dilated congestive cardiomyopathy,restrictive cardiomyopathy, valvular heart diseases, endocarditis,pericardial disease, cardiac tumors, aortic and peripheral aneurysms,aortic dissection, inflammation of the aorta, occlusion of the abdominalaorta and its branches, peripheral vascular disorders, occlusivearterial disorders, peripheral atherosclerotic disease, thromboangitisobliterans, functional peripheral arterial disorders, Raynaud'sphenomenon and disease, acrocyanosis, erythromelalgia, venous diseases,venous thrombosis, varicose veins, arteriovenous fistula, lymphedema,lipedema, unstable angina, reperfusion injury, post pump syndrome,ischemia-reperfusion injury, and the like. Such a method can optionallycomprise administering an effective amount of a composition orpharmaceutical composition comprising at least one anti-TNF antibody toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy.

The present invention also provides a method for modulating or treatingat least one infectious disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: acute orchronic bacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis (A, B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, E. coli0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,Mycobacterium tuberculosis, Mycobacterium avium intracellulare,Pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, viral-associated hemaphagocytic syndrome, vitalencephalitis/aseptic meningitis, and the like.

The present invention also provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: leukemia, acuteleukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL,acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chroniclymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome(MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma,non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi'ssarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngealcarcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas,sarcomas, malignant melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer related bone pain, and the like.

The present invention also provides a method for modulating or treatingat least one neurologic disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of:neurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders, suchas lesions of the corticospinal system; disorders of the basal gangliaor cerebellar disorders; hyperkinetic movement disorders such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo Palsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasiaa, andmitochondrial multisystem disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit′ such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,and the like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one TNF antibody or specified portion or variant toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy. See, e.g., the Merck Manual, 16^(th) Edition,Merck & Company, Rahway, N.J. (1992)

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-TNF antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy. Sucha method can optionally further comprise co-administration orcombination therapy for treating such immune diseases, wherein theadministering of said at least one anti-TNF antibody, specified portionor variant thereof, further comprises administering, beforeconcurrently, and/or after, at least one selected from at least one TNFantagonist (e.g., but not limited to a TNF antibody or fragment, asoluble TNF receptor or fragment, fusion proteins thereof, or a smallmolecule TNF antagonist), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a flurorquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which references are entirely incorporatedherein by reference.

TNF antagonists suitable for compositions, combination therapy,co-administration, devices and/or methods of the present invention(further comprising at least one anti body, specified portion andvariant thereof, of the present invention), include, but are not limitedto, anti-TNF antibodies, antigen-binding fragments thereof, and receptormolecules which bind specifically to TNF; compounds which prevent and/orinhibit TNF synthesis, TNF release or its action on target cells, suchas thalidomide, tenidap, phosphodiesterase inhibitors (e.g.,pentoxifylline and rolipram), A2b adenosine receptor agonists and A2badenosine receptor enhancers; compounds which prevent and/or inhibit TNFreceptor signaling, such as mitogen activated protein (MAP) kinaseinhibitors; compounds which block and/or inhibit membrane TNF cleavage,such as metalloproteinase inhibitors; compounds which block and/orinhibit TNF activity, such as angiotensin converting enzyme (ACE)inhibitors (e.g., captopril); and compounds which block and/or inhibitTNF production and/or synthesis, such as MAP kinase inhibitors.

As used herein, a “tumor necrosis factor antibody,” “TNF antibody,”“TNFα antibody,” or fragment and the like decreases, blocks, inhibits,abrogates or interferes with TNFα activity in vitro, in situ and/orpreferably in vivo. For example, a suitable TNF human antibody of thepresent invention can bind TNFα and includes anti-TNF antibodies,antigen-binding fragments thereof, and specified mutants or domainsthereof that bind specifically to TNFα. A suitable TNF antibody orfragment can also decrease block, abrogate, interfere, prevent and/orinhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptorsignaling, membrane TNF cleavage, TNF activity, TNF production and/orsynthesis.

Chimeric antibody cA2 consists of the antigen binding variable region ofthe high-affinity neutralizing mouse anti-human TNFα IgG1 antibody,designated A2, and the constant regions of a human IgG1, kappaimmunoglobulin. The human IgG1 Fc region improves allogeneic antibodyeffector function, increases the circulating serum half-life anddecreases the immunogenicity of the antibody. The avidity and epitopespecificity of the chimeric antibody cA2 is derived from the variableregion of the murine antibody A2. In a particular embodiment, apreferred source for nucleic acids encoding the variable region of themurine antibody A2 is the A2 hybridoma cell line.

Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural andrecombinant human TNFα in a dose dependent manner. From binding assaysof chimeric antibody cA2 and recombinant human TNFα, the affinityconstant of chimeric antibody cA2 was calculated to be 1.04×10¹⁰ M⁻¹.Preferred methods for determining monoclonal antibody specificity andaffinity by competitive inhibition can be found in Harlow, et al.,antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988; Colligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, NewYork, (1992-2000); Kozbor et al., Immunol. Today, 4:72-79 (1983);Ausubel et al., eds. Current Protocols in Molecular Biology, WileyInterscience, New York (1987-2000); and Muller, Meth. Enzymol.,92:589-601 (1983), which references are entirely incorporated herein byreference.

In a particular embodiment, murine monoclonal antibody A2 is produced bya cell line designated c134A. Chimeric antibody cA2 is produced by acell line designated c168A.

Additional examples of monoclonal anti-TNF antibodies that can be usedin the present invention are described in the art (see, e.g., U.S. Pat.No. 5,231,024; Möller, A. et al., Cytokine 2(3):162-169 (1990); U.S.application Ser. No. 07/943,852 (filed Sep. 11, 1992); Rathjen et al.,International Publication No. WO 91/02078 (published Feb. 21, 1991);Rubin et al., EPO Patent Publication No. 0 218 868 (published Apr. 22,1987); Yone et al., EPO Patent Publication No. 0 288 088 (Oct. 26,1988); Liang, et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986);Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma6:359-369 (1987); Bringman, et al., Hybridoma 6:489-507 (1987); andHirai, et al., J. Immunol. Meth. 96:57-62 (1987), which references areentirely incorporated herein by reference).

TNF Receptor Molecules.

Preferred TNF receptor molecules useful in the present invention arethose that bind TNFα with high affinity (see, e.g., Feldmann et al.,International Publication No. WO 92/07076 (published Apr. 30, 1992);Schall et al., Cell 61:361-370 (1990); and Loetscher et al., Cell61:351-359 (1990), which references are entirely incorporated herein byreference) and optionally possess low immunogenicity. In particular, the55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptorsare useful in the present invention. Truncated forms of these receptors,comprising the extracellular domains (ECD) of the receptors orfunctional portions thereof (see, e.g., Corcoran et al., Eur. J.Biochem. 223:831-840 (1994)), are also useful in the present invention.Truncated forms of the TNF receptors, comprising the ECD, have beendetected in urine and serum as 30 kDa and 40 kDa TNFα inhibitory bindingproteins (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)).TNF receptor multimeric molecules and TNF immunoreceptor fusionmolecules, and derivatives and fragments or portions thereof, areadditional examples of TNF receptor molecules which are useful in themethods and compositions of the present invention. The TNF receptormolecules which can be used in the invention are characterized by theirability to treat patients for extended periods with good to excellentalleviation of symptoms and low toxicity. Low immunogenicity and/or highaffinity, as well as other undefined properties, can contribute to thetherapeutic results achieved.

TNF receptor multimeric molecules useful in the present inventioncomprise all or a functional portion of the ECD of two or more TNFreceptors linked via one or more polypeptide linkers or other nonpeptidelinkers, such as polyethylene glycol (PEG). The multimeric molecules canfurther comprise a signal peptide of a secreted protein to directexpression of the multimeric molecule. These multimeric molecules andmethods for their production have been described in U.S. applicationSer. No. 08/437,533 (filed May 9, 1995), the content of which isentirely incorporated herein by reference.

TNF immunoreceptor fusion molecules useful in the methods andcompositions of the present invention comprise at least one portion ofone or more immunoglobulin molecules and all or a functional portion ofone or more TNF receptors. These immunoreceptor fusion molecules can beassembled as monomers, or hetero- or homo-multimers. The immunoreceptorfusion molecules can also be monovalent or multivalent. An example ofsuch a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusionprotein. TNF immunoreceptor fusion molecules and methods for theirproduction have been described in the art (Lesslauer et al., Eur. J.Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci.USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994);Butler et al., Cytokine 6(6):616-623 (1994); Baker et al., Eur. J.Immunol. 24:2040-2048 (1994); Beutler et al., U.S. Pat. No. 5,447,851;and U.S. application Ser. No. 08/442,133 (filed May 16, 1995), each ofwhich references are entirely incorporated herein by reference). Methodsfor producing immunoreceptor fusion molecules can also be found in Caponet al., U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538;and Capon et al., Nature 337:525-531 (1989), which references areentirely incorporated herein by reference.

A functional equivalent, derivative, fragment or region of TNF receptormolecule refers to the portion of the TNF receptor molecule, or theportion of the TNF receptor molecule sequence which encodes TNF receptormolecule, that is of sufficient size and sequences to functionallyresemble TNF receptor molecules that can be used in the presentinvention (e.g., bind TNFα with high affinity and possess lowimmunogenicity). A functional equivalent of TNF receptor molecule alsoincludes modified TNF receptor molecules that functionally resemble TNFreceptor molecules that can be used in the present invention (e.g., bindTNFα with high affinity and possess low immunogenicity). For example, afunctional equivalent of TNF receptor molecule can contain a “SILENT”codon or one or more amino acid substitutions, deletions or additions(e.g., substitution of one acidic amino acid for another acidic aminoacid; or substitution of one codon encoding the same or differenthydrophobic amino acid for another codon encoding a hydrophobic aminoacid). See Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2000).

Cytokines include any known cytokine. See, e.g., CopewithCytokines.com.Cytokine antagonists include, but are not limited to, any antibody,fragment or mimetic, any soluble receptor, fragment or mimetic, anysmall molecule antagonist, or any combination thereof.

Therapeutic Treatments.

Any method of the present invention can comprise a method for treating aTNF mediated disorder, comprising administering an effective amount of acomposition or pharmaceutical composition comprising at least oneanti-TNF antibody to a cell, tissue, organ, animal or patient in need ofsuch modulation, treatment or therapy. Such a method can optionallyfurther comprise co-administration or combination therapy for treatingsuch immune diseases, wherein the administering of said at least oneanti-TNF antibody, specified portion or variant thereof, furthercomprises administering, before concurrently, and/or after, at least oneselected from at least one TNF antagonist (e.g., but not limited to aTNF antibody or fragment, a soluble TNF receptor or fragment, fusionproteins thereof, or a small molecule TNF antagonist), an antirheumatic(e.g., methotrexate, auranofin, aurothioglucose, azathioprine,etanercept, gold sodium thiomalate, hydroxychloroquine sulfate,leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anethetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin,a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic,a corticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist.

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one anti-TNFantibody composition that total, on average, a range from at least about0.01 to 500 milligrams of at least one anti-TNF antibody per kilogram ofpatient per dose, and preferably from at least about 0.1 to 100milligrams antibody/kilogram of patient per single or multipleadministration, depending upon the specific activity of contained in thecomposition. Alternatively, the effective serum concentration cancomprise 0.1-5000 μg/ml serum concentration per single or multipleadministration. Suitable dosages are known to medical practitioners andwill, of course, depend upon the particular disease state, specificactivity of the composition being administered, and the particularpatient undergoing treatment. In some instances, to achieve the desiredtherapeutic amount, it can be necessary to provide for repeatedadministration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 15, 15.5,15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9,20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and/or 5000μg/ml serum concentration per single or multiple administration, or anyrange, value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to 10milligrams per kilogram per administration or in sustained release formis effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or alternatively or additionally, at least one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20years, or any combination thereof, using single, infusion or repeateddoses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion or lyophilized powder in association, orseparately provided, with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 1-10% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by known or suitabletechniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Alternative Administration.

Many known and developed modes of administration can be used accordingto the present invention for administering pharmaceutically effectiveamounts of at least one anti-TNF antibody according to the presentinvention. While pulmonary administration is used in the followingdescription, other modes of administration can be used according to thepresent invention with suitable results.

TNF antibodies of the present invention can be delivered in a carrier,as a solution, emulsion, colloid, or suspension, or as a dry powder,using any of a variety of devices and methods suitable foradministration by inhalation or other modes described here within orknown in the art.

Parenteral Formulations and Administration.

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent such as aqueous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthetic mono- or di- or tri-glycerides. Parental administration isknown in the art and includes, but is not limited to, conventional meansof injections, a gas pressured needle-less injection device as describedin U.S. Pat. No. 5,851,198, and a laser perforator device as describedin U.S. Pat. No. 5,839,446 entirely incorporated herein by reference.

Alternative Delivery.

The invention further relates to the administration of at least oneanti-TNF antibody by parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.At least one anti-TNF antibody composition can be prepared for use forparenteral (subcutaneous, intramuscular or intravenous) or any otheradministration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms such as, but not limited to, creams and suppositories;for buccal, or sublingual administration such as, but not limited to, inthe form of tablets or capsules; or intranasally such as, but notlimited to, the form of powders, nasal drops or aerosols or certainagents; or transdermally such as not limited to a gel, ointment, lotion,suspension or patch delivery system with chemical enhancers such asdimethyl sulfoxide to either modify the skin structure or to increasethe drug concentration in the transdermal patch (Junginger, et al. In“Drug Permeation Enhancement”; Hsieh, D. S., Eds., pp. 59-90 (MarcelDekker, Inc. New York 1994, entirely incorporated herein by reference),or with oxidizing agents that enable the application of formulationscontaining proteins and peptides onto the skin (WO 98/53847), orapplications of electric fields to create transient transport pathwayssuch as electroporation, or to increase the mobility of charged drugsthrough the skin such as iontophoresis, or application of ultrasoundsuch as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the abovepublications and patents being entirely incorporated herein byreference).

Pulmonary/Nasal Administration.

For pulmonary administration, preferably at least one anti-TNF antibodycomposition is delivered in a particle size effective for reaching thelower airways of the lung or sinuses. According to the invention, atleast one anti-TNF antibody can be delivered by any of a variety ofinhalation or nasal devices known in the art for administration of atherapeutic agent by inhalation. These devices capable of depositingaerosolized formulations in the sinus cavity or alveoli of a patientinclude metered dose inhalers, nebulizers, dry powder generators,sprayers, and the like. Other devices suitable for directing thepulmonary or nasal administration of antibodies are also known in theart. All such devices can use of formulations suitable for theadministration for the dispensing of antibody in an aerosol. Suchaerosols can be comprised of either solution (both aqueous andnon-aqueous) or solid particles. Metered dose inhalers like theVentolin® metered dose inhaler, typically use a propellant gas andrequire actuation during inspiration (See, e.g., WO 94/16970, WO98/35888). Dry powder inhalers like Turbuhaler™ (Astra), Rotahaler®(Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura), devices marketed byInhale Therapeutics, and the Spinhaler® powder inhaler (Fisons), usebreath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No.5,458,135 Inhale, WO 94/06498 Fisons, entirely incorporated herein byreference). Nebulizers like AERx™ Aradigm, the Ultravent® nebulizer(Mallinckrodt), and the Acorn II® nebulizer (Marquest Medical Products)(U.S. Pat. No. 5,404,871 Aradigm, WO 97/22376), the above referencesentirely incorporated herein by reference, produce aerosols fromsolutions, while metered dose inhalers, dry powder inhalers, etc.generate small particle aerosols. These specific examples ofcommercially available inhalation devices are intended to be arepresentative of specific devices suitable for the practice of thisinvention and are not intended as limiting the scope of the invention.Preferably, a composition comprising at least one anti-TNF antibody isdelivered by a dry powder inhaler or a sprayer. There are a severaldesirable features of an inhalation device for administering at leastone antibody of the present invention. For example, delivery by theinhalation device is advantageously reliable, reproducible, andaccurate. The inhalation device can optionally deliver small dryparticles, e.g. less than about 10 μm, preferably about 1-5 μm, for goodrespirability.

Administration of TNF Antibody Compositions as a Spray.

A spray including TNF antibody composition protein can be produced byforcing a suspension or solution of at least one anti-TNF antibodythrough a nozzle under pressure. The nozzle size and configuration, theapplied pressure, and the liquid feed rate can be chosen to achieve thedesired output and particle size. An electrospray can be produced, forexample, by an electric field in connection with a capillary or nozzlefeed. Advantageously, particles of at least one anti-TNF antibodycomposition protein delivered by a sprayer have a particle size lessthan about 10 μm, preferably in the range of about 1 μm to about 5 μm,and most preferably about 2 μm to about 3 μm.

Formulations of at least one anti-TNF antibody composition proteinsuitable for use with a sprayer typically include antibody compositionprotein in an aqueous solution at a concentration of about 0.1 mg toabout 100 mg of at least one anti-TNF antibody composition protein perml of solution or mg/gm, or any range or value therein, e.g., but notlimited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml ormg/gm. The formulation can include agents such as an excipient, abuffer, an isotonicity agent, a preservative, a surfactant, and,preferably, zinc. The formulation can also include an excipient or agentfor stabilization of the antibody composition protein, such as a buffer,a reducing agent, a bulk protein, or a carbohydrate. Bulk proteinsuseful in formulating antibody composition proteins include albumin,protamine, or the like. Typical carbohydrates useful in formulatingantibody composition proteins include sucrose, mannitol, lactose,trehalose, glucose, or the like. The antibody composition proteinformulation can also include a surfactant, which can reduce or preventsurface-induced aggregation of the antibody composition protein causedby atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbitol fatty acidesters. Amounts will generally range between 0.001 and 14% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan monooleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein such as TNF antibodies, or specified portionsor variants, can also be included in the formulation.

Administration of TNF Antibody Compositions by a Nebulizer.

Antibody composition protein can be administered by a nebulizer, such asjet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer,a compressed air source is used to create a high-velocity air jetthrough an orifice. As the gas expands beyond the nozzle, a low-pressureregion is created, which draws a solution of antibody compositionprotein through a capillary tube connected to a liquid reservoir. Theliquid stream from the capillary tube is sheared into unstable filamentsand droplets as it exits the tube, creating the aerosol. A range ofconfigurations, flow rates, and baffle types can be employed to achievethe desired performance characteristics from a given jet nebulizer. Inan ultrasonic nebulizer, high-frequency electrical energy is used tocreate vibrational, mechanical energy, typically employing apiezoelectric transducer. This energy is transmitted to the formulationof antibody composition protein either directly or through a couplingfluid, creating an aerosol including the antibody composition protein.Advantageously, particles of antibody composition protein delivered by anebulizer have a particle size less than about 10 μm, preferably in therange of about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm.

Formulations of at least one anti-TNF antibody suitable for use with anebulizer, either jet or ultrasonic, typically include a concentrationof about 0.1 mg to about 100 mg of at least one anti-TNF antibodyprotein per ml of solution. The formulation can include agents such asan excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the at least one anti-TNFantibody composition protein, such as a buffer, a reducing agent, a bulkprotein, or a carbohydrate. Bulk proteins useful in formulating at leastone anti-TNF antibody composition proteins include albumin, protamine,or the like. Typical carbohydrates useful in formulating at least oneanti-TNF antibody include sucrose, mannitol, lactose, trehalose,glucose, or the like. The at least one anti-TNF antibody formulation canalso include a surfactant, which can reduce or prevent surface-inducedaggregation of the at least one anti-TNF antibody caused by atomizationof the solution in forming an aerosol. Various conventional surfactantscan be employed, such as polyoxyethylene fatty acid esters and alcohols,and polyoxyethylene sorbital fatty acid esters. Amounts will generallyrange between 0.001 and 4% by weight of the formulation. Especiallypreferred surfactants for purposes of this invention are polyoxyethylenesorbitan mono-oleate, polysorbate 80, polysorbate 20, or the like.Additional agents known in the art for formulation of a protein such asantibody protein can also be included in the formulation.

Administration of TNF Antibody Compositions by a Metered Dose Inhaler.

In a metered dose inhaler (MDI), a propellant, at least one anti-TNFantibody, and any excipients or other additives are contained in acanister as a mixture including a liquefied compressed gas. Actuation ofthe metering valve releases the mixture as an aerosol, preferablycontaining particles in the size range of less than about 10 μm,preferably about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm. The desired aerosol particle size can be obtained byemploying a formulation of antibody composition protein produced byvarious methods known to those of skill in the art, includingjet-milling, spray drying, critical point condensation, or the like.Preferred metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant.

Formulations of at least one anti-TNF antibody for use with ametered-dose inhaler device will generally include a finely dividedpowder containing at least one anti-TNF antibody as a suspension in anon-aqueous medium, for example, suspended in a propellant with the aidof a surfactant. The propellant can be any conventional materialemployed for this purpose, such as chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.Preferably the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one anti-TNF antibody as a suspensionin the propellant, to protect the active agent against chemicaldegradation, and the like. Suitable surfactants include sorbitantrioleate, soya lecithin, oleic acid, or the like. In some cases,solution aerosols are preferred using solvents such as ethanol.Additional agents known in the art for formulation of a protein can alsobe included in the formulation.

One of ordinary skill in the art will recognize that the methods of thecurrent invention can be achieved by pulmonary administration of atleast one anti-TNF antibody compositions via devices not describedherein.

Oral Formulations and Administration.

Formulations for oral rely on the co-administration of adjuvants (e.g.,resorcinols and nonionic surfactants such as polyoxyethylene oleyl etherand n-hexadecylpolyethylene ether) to increase artificially thepermeability of the intestinal walls, as well as the co-administrationof enzymatic inhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation. The active constituent compound of the solid-type dosageform for oral administration can be mixed with at least one additive,including sucrose, lactose, cellulose, mannitol, trehalose, raffinose,maltitol, dextran, starches, agar, arginates, chitins, chitosans,pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin,synthetic or semisynthetic polymer, and glyceride. These dosage formscan also contain other type(s) of additives, e.g., inactive dilutingagent, lubricant such as magnesium stearate, paraben, preserving agentsuch as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidant suchas cysteine, disintegrator, binder, thickener, buffering agent,sweetening agent, flavoring agent, perfuming agent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. Nos. 5,879,681 and 5,5,871,753 are usedto deliver biologically active agents orally are known in the art.

Mucosal Formulations and Administration.

For absorption through mucosal surfaces, compositions and methods ofadministering at least one anti-TNF antibody include an emulsioncomprising a plurality of submicron particles, a mucoadhesivemacromolecule, a bioactive peptide, and an aqueous continuous phase,which promotes absorption through mucosal surfaces by achievingmucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucoussurfaces suitable for application of the emulsions of the presentinvention can include corneal, conjunctival, buccal, sublingual, nasal,vaginal, pulmonary, stomachic, intestinal, and rectal routes ofadministration. Formulations for vaginal or rectal administration, e.g.suppositories, can contain as excipients, for example,polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulationsfor intranasal administration can be solid and contain as excipients,for example, lactose or can be aqueous or oily solutions of nasal drops.For buccal administration excipients include sugars, calcium stearate,magnesium stearate, pregelinatined starch, and the like (U.S. Pat. No.5,849,695).

Transdermal Formulations and Administration.

For transdermal administration, the at least one anti-TNF antibody isencapsulated in a delivery device such as a liposome or polymericnanoparticles, microparticle, microcapsule, or microspheres (referred tocollectively as microparticles unless otherwise stated). A number ofsuitable devices are known, including microparticles made of syntheticpolymers such as polyhydroxy acids such as polylactic acid, polyglycolicacid and copolymers thereof, polyorthoesters, polyanhydrides, andpolyphosphazenes, and natural polymers such as collagen, polyaminoacids, albumin and other proteins, alginate and other polysaccharides,and combinations thereof (U.S. Pat. No. 5,814,599).

Prolonged Administration and Formulations.

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot or implant dosage forms can be utilized. Forexample, a dosage form can contain a pharmaceutically acceptablenon-toxic salt of the compounds that has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) asalt with a polyvalent metal cation such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and thelike, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g., sesame oil, suitable forinjection. Particularly preferred salts are zinc salts, zinc tannatesalts, pamoate salts, and the like. Another type of slow release depotformulation for injection would contain the compound or salt dispersedfor encapsulated in a slow degrading, non-toxic, non-antigenic polymersuch as a polylactic acid/polyglycolic acid polymer for example asdescribed in U.S. Pat. No. 3,773,919. The compounds or, preferably,relatively insoluble salts such as those described above can also beformulated in cholesterol matrix silastic pellets, particularly for usein animals. Additional slow release, depot or implant formulations, e.g.gas or liquid liposomes are known in the literature (U.S. Pat. No.5,770,222 and “Sustained and Controlled Release Drug Delivery Systems”,J. R. Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Example 1: Cloning and Expression of TNF Antibody in Mammalian Cells

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, theantibody coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pIRES1neo, pRetro-Off,pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL andPMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC® 37152), pSV2dhfr(ATCC® 37146) and pBC12MI. Mammalian host cells that could be usedinclude human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

Cloning and Expression in CHO Cells

One vector commonly used for expression in CHO cells is pC4. Plasmid pC4is a derivative of the plasmid pSV2-dhfr (ATCC® 37146). The plasmidcontains the mouse DHFR gene under control of the SV40 early promoter.Chinese hamster ovary cells or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (e.g., alpha minus MEM, LifeTechnologies, Gaithersburg, Md.) supplemented with the chemotherapeuticagent methotrexate. The amplification of the DHFR genes in cellsresistant to methotrexate (MTX) has been well documented (see, e.g., F.W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C.Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M.A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasingconcentrations of MTX develop resistance to the drug by overproducingthe target enzyme, DHFR, as a result of amplification of the DHFR gene.If a second gene is linked to the DHFR gene, it is usually co-amplifiedand over-expressed. It is known in the art that this approach can beused to develop cell lines carrying more than 1,000 copies of theamplified gene(s). Subsequently, when the methotrexate is withdrawn,cell lines are obtained that contain the amplified gene integrated intoone or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human beta-actin promoter,the SV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express the TNF ina regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc.Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For the polyadenylation ofthe mRNA other signals, e.g., from the human growth hormone or globingenes can be used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed, and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary (CHO) cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin. The plasmidpSV2neo contains a dominant selectable marker, the neo gene from Tn5encoding an enzyme that confers resistance to a group of antibioticsincluding G418. The cells are seeded in alpha minus MEM supplementedwith 1 μg/ml G418. After 2 days, the cells are trypsinized and seeded inhybridoma cloning plates (Greiner, Germany) in alpha minus MEMsupplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 μg/ml G418.After about 10-14 days single clones are trypsinized and then seeded in6-well petri dishes or 10 ml flasks using different concentrations ofmethotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing atthe highest concentrations of methotrexate are then transferred to new6-well plates containing even higher concentrations of methotrexate (1mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated untilclones are obtained that grow at a concentration of 100-200 mM.Expression of the desired gene product is analyzed, for instance, bySDS-PAGE and Western blot or by reverse phase HPLC analysis.

Example 2: Generation of High Affinity Human IgG Monoclonal AntibodiesReactive with Human TNF Using Transgenic Mice

Summary.

Transgenic mice have been used that contain human heavy and light chainimmunoglobulin genes to generate high affinity, completely human,monoclonal antibodies that can be used therapeutically to inhibit theaction of TNF for the treatment of one or more TNF-mediated disease.(CBA/J×C57/BL6/J) F2 hybrid mice containing human variable and constantregion antibody transgenes for both heavy and light chains are immunizedwith human recombinant TNF (Taylor et al., Intl. Immunol. 6:579-591(1993); Lonberg, et al., Nature 368:856-859 (1994); Neuberger, M.,Nature Biotech. 14:826 (1996); Fishwild, et al., Nature Biotechnology14:845-851 (1996)). Several fusions yielded one or more panels ofcompletely human TNF reactive IgG monoclonal antibodies. The completelyhuman anti-TNF antibodies are further characterized. All are IgG1κ. Suchantibodies are found to have affinity constants somewhere between 1×10⁹and 9×10¹². The unexpectedly high affinities of these fully humanmonoclonal antibodies make them suitable candidates for therapeuticapplications in TNF related diseases, pathologies or disorders.

ABBREVIATIONS

BSA—bovine serum albumin; CO₂—carbon dioxide; DMSO—dimethyl sulfoxide;EIA—enzyme immunoassay; FBS—fetal bovine serum; H₂O₂—hydrogen peroxide;HRP—horseradish peroxidase; ID—interadermal; Ig—immunoglobulin;TNF—tissue necrosis factor alpha; IP—intraperitoneal; IV—intravenous;Mab or mAb—monoclonal antibody; OD—optical density;OPD—o-Phenylenediamine dihydrochloride; PEG—polyethylene glycol;PSA—penicillin, streptomycin, amphotericin; RT—room temperature;SQ—subcutaneous; v/v—volume per volume; w/v—weight per volume.

Materials and Methods

Animals.

Transgenic mice that can express human antibodies are known in the art(and are commercially available (e.g., from GenPharm International, SanJose, Calif.; Abgenix, Freemont, Calif., and others) that express humanimmunoglobulins but not mouse IgM or Igκ. For example, such transgenicmice contain human sequence transgenes that undergo V(D)J joining,heavy-chain class switching, and somatic mutation to generate arepertoire of human sequence immunoglobulins (Lonberg, et al., Nature368:856-859 (1994)). The light chain transgene can be derived, e.g., inpart from a yeast artificial chromosome clone that includes nearly halfof the germline human Vκ region. In addition, the heavy-chain transgenecan encode both human μ and human γ1 (Fishwild, et al., NatureBiotechnology 14:845-851 (1996)) and/or γ3 constant regions. Micederived from appropriate genotypic lineages can be used in theimmunization and fusion processes to generate fully human monoclonalantibodies to TNF.

Immunization.

One or more immunization schedules can be used to generate the anti-TNFhuman hybridomas. The first several fusions can be performed after thefollowing exemplary immunization protocol, but other similar knownprotocols can be used. Several 14-20 week old female and/or surgicallycastrated transgenic male mice are immunized IP and/or ID with 1-1000 μgof recombinant human TNF emulsified with an equal volume of TITERMAX orcomplete Freund's adjuvant in a final volume of 100-4004 (e.g., 200).Each mouse can also optionally receive 1-10 μg in 100 μL physiologicalsaline at each of 2 SQ sites. The mice can then be immunized 1-7, 5-12,10-18, 17-25 and/or 21-34 days later IP (1-400 μg) and SQ (1-400 μg×2)with TNF emulsified with an equal volume of TITERMAX or incompleteFreund's adjuvant. Mice can be bled 12-25 and 25-40 days later byretro-orbital puncture without anti-coagulant. The blood is then allowedto clot at RT for one hour and the serum is collected and titered usingan TNF EIA assay according to known methods. Fusions are performed whenrepeated injections do not cause titers to increase. At that time, themice can be given a final IV booster injection of 1-400 μg TNF dilutedin 100 μL physiological saline. Three days later, the mice can beeuthanized by cervical dislocation and the spleens removed asepticallyand immersed in 10 mL of cold phosphate buffered saline (PBS) containing100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericinB (PSA). The splenocytes are harvested by sterilely perfusing the spleenwith PSA-PBS. The cells are washed once in cold PSA-PBS, counted usingTrypan blue dye exclusion and resuspended in RPMI 1640 media containing25 mM Hepes.

Cell Fusion.

Fusion can be carried out at a 1:1 to 1:10 ratio of murine myeloma cellsto viable spleen cells according to known methods, e.g., as known in theart. As a non-limiting example, spleen cells and myeloma cells can bepelleted together. The pellet can then be slowly resuspended, over 30seconds, in 1 mL of 50% (w/v) PEG/PBS solution (PEG molecular weight1,450, Sigma) at 37° C. The fusion can then be stopped by slowly adding10.5 mL of RPMI 1640 medium containing 25 mM Hepes (37° C.) over 1minute. The fused cells are centrifuged for 5 minutes at 500-1500 rpm.The cells are then resuspended in HAT medium (RPMI 1640 mediumcontaining 25 mM Hepes, 10% Fetal Clone I serum (Hyclone), 1 mM sodiumpyruvate, 4 mM L-glutamine, 10 μg/mL gentamicin, 2.5% Origen culturingsupplement (Fisher), 10% 653-conditioned RPMI 1640/Hepes media, 50 μM2-mercaptoethanol, 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μMthymidine) and then plated at 200 μL/well in fifteen 96-well flat bottomtissue culture plates. The plates are then placed in a humidified 37° C.incubator containing 5% CO₂ and 95% air for 7-10 days.

Detection of Human IgG Anti-TNF Antibodies in Mouse Serum.

Solid phase EIA's can be used to screen mouse sera for human IgGantibodies specific for human TNF. Briefly, plates can be coated withTNF at 2 μg/mL in PBS overnight. After washing in 0.15M salinecontaining 0.02% (v/v) Tween 20, the wells can be blocked with 1% (w/v)BSA in PBS, 200 μL/well for 1 hour at RT. Plates are used immediately orfrozen at −20° C. for future use. Mouse serum dilutions are incubated onthe TNF coated plates at 50 μL/well at RT for 1 hour. The plates arewashed and then probed with 50 μL/well HRP-labeled goat anti-human IgG,Fc specific diluted 1:30,000 in 1% BSA-PBS for 1 hour at RT. The platescan again be washed and 100 μL/well of the citrate-phosphate substratesolution (0.1M citric acid and 0.2M sodium phosphate, 0.01% H₂O₂ and 1mg/mL OPD) is added for 15 minutes at RT. Stop solution (4N sulfuricacid) is then added at 25 μL/well and the OD's are read at 490 nm via anautomated plate spectrophotometer.

Detection of Completely Human Immunoglobulins in Hybridoma Supernates.

Growth positive hybridomas secreting fully human immunoglobulins can bedetected using a suitable EIA. Briefly, 96 well pop-out plates (VWR,610744) can be coated with 10 μg/mL goat anti-human IgG Fc in sodiumcarbonate buffer overnight at 4° C. The plates are washed and blockedwith 1% BSA-PBS for one hour at 37° C. and used immediately or frozen at−20° C. Undiluted hybridoma supernatants are incubated on the plates forone hour at 37° C. The plates are washed and probed with HRP labeledgoat anti-human kappa diluted 1:10,000 in 1% BSA-PBS for one hour at 37°C. The plates are then incubated with substrate solution as describedabove.

Determination of Fully Human Anti-TNF Reactivity.

Hybridomas, as above, can be simultaneously assayed for reactivity toTNF using a suitable RIA or other assay. For example, supernatants areincubated on goat anti-human IgG Fc plates as above, washed and thenprobed with radiolabled TNF with appropriate counts per well for 1 hourat RT. The wells are washed twice with PBS and bound radiolabled TNF isquantitated using a suitable counter.

Human IgG1κ anti-TNF secreting hybridomas can be expanded in cellculture and serially subcloned by limiting dilution. The resultingclonal populations can be expanded and cryopreserved in freezing medium(95% FBS, 5% DMSO) and stored in liquid nitrogen.

Isotyping.

Isotype determination of the antibodies can be accomplished using an EIAin a format similar to that used to screen the mouse immune sera forspecific titers. TNF can be coated on 96-well plates as described aboveand purified antibody at 2 μg/mL can be incubated on the plate for onehour at RT. The plate is washed and probed with HRP labeled goatanti-human IgG₁ or HRP labeled goat anti-human IgG3 diluted at 1:4000 in1% BSA-PBS for one hour at RT. The plate is again washed and incubatedwith substrate solution as described above.

Binding Kinetics of Human Anti-Human TNF Antibodies with Human TNF.

Binding characteristics for antibodies can be suitably assessed using anTNF capture EIA and BIAcore technology, for example. Gradedconcentrations of purified human TNF antibodies can be assessed forbinding to EIA plates coated with 2 μg/mL of TNF in assays as describedabove. The OD's can be then presented as semi-log plots showing relativebinding efficiencies.

Quantitative binding constants can be obtained, e.g., as follows, or byany other known suitable method. A BIAcore CM-5 (carboxymethyl) chip isplaced in a BIAcore 2000 unit. HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.005% v/v P20 surfactant, pH 7.4) is flowed over a flow cellof the chip at 5 μL/minute until a stable baseline is obtained. Asolution (100 μL) of 15 mg of EDC(N-ethyl-N′-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200μL water is added to 100 μL of a solution of 2.3 mg of NHS(N-hydroxysuccinimide) in 200 μL water. Forty (40) μL of the resultingsolution is injected onto the chip. Six μL of a solution of human TNF(15 μg/mL in 10 mM sodium acetate, pH 4.8) is injected onto the chip,resulting in an increase of ca. 500 RU. The buffer is changed toTBS/Ca/Mg/BSA running buffer (20 mM Tris, 0.15 M sodium chloride, 2 mMcalcium chloride, 2 mM magnesium acetate, 0.5% Triton X-100, 25 μg/mLBSA, pH 7.4) and flowed over the chip overnight to equilibrate it and tohydrolyze or cap any unreacted succinimide esters.

Antibodies are dissolved in the running buffer at 33.33, 16.67, 8.33,and 4.17 nM. The flow rate is adjusted to 30 μL/min and the instrumenttemperature to 25° C. Two flow cells are used for the kinetic runs, oneon which TNF had been immobilized (sample) and a second, underivatizedflow cell (blank). 1204 of each antibody concentration is injected overthe flow cells at 30 μL/min (association phase) followed by anuninterrupted 360 seconds of buffer flow (dissociation phase). Thesurface of the chip is regenerated (tissue necrosis factoralpha/antibody complex dissociated) by two sequential injections of 30μL each of 2 M guanidine thiocyanate.

Analysis of the data is done using BIA evaluation 3.0 or CLAMP 2.0, asknown in the art. For each antibody concentration the blank sensogram issubtracted from the sample sensogram. A global fit is done for bothdissociation (k_(d), sec⁻) and association (k_(a), mol⁻¹ sec⁻¹) and thedissociation constant (K_(D), mol) calculated (k_(d)/k_(a)). Where theantibody affinity is high enough that the RUs of antibody capturedare >100, additional dilutions of the antibody are run.

Results and Discussion

Generation of Anti-Human TNF Monoclonal Antibodies.

Several fusions are performed, and each fusion is seeded in 15 plates(1440 wells/fusion) that yield several dozen antibodies specific forhuman TNF. Of these, some are found to consist of a combination of humanand mouse Ig chains. The remaining hybridomas secret anti-TNF antibodiesconsisting solely of human heavy and light chains. Of the humanhybridomas all are expected to be IgG1κ.

Binding Kinetics of Human Anti-Human TNF Antibodies.

ELISA analysis confirms that purified antibody from most or all of thesehybridomas bind TNF in a concentration-dependent manner. FIG. 1 and FIG.2 show the results of the relative binding efficiency of theseantibodies. In this case, the avidity of the antibody for its cognateantigen (epitope) is measured. It should be noted that binding TNFdirectly to the EIA plate can cause denaturation of the protein and theapparent binding affinities cannot be reflective of binding toundenatured protein. Fifty percent binding is found over a range ofconcentrations.

Quantitative binding constants are obtained using BIAcore analysis ofthe human antibodies and reveals that several of the human monoclonalantibodies are very high affinity with K_(D) in the range of 1×10⁻⁹ to7×10⁻¹².

Conclusions.

Several fusions are performed utilizing splenocytes from hybrid micecontaining human variable and constant region antibody transgenes thatare immunized with human TNF. A set of several completely human TNFreactive IgG monoclonal antibodies of the IgG1κ isotype were generated.The completely human anti-TNF antibodies are further characterized.Several of generated antibodies have affinity constants between 1×10⁹and 9×10¹². The unexpectedly high affinities of these fully humanmonoclonal antibodies make them suitable for therapeutic applications inTNF-dependent diseases, pathologies or related conditions.

Example 3: Generation of Human IgG Monoclonal Antibodies Reactive toHuman TNFα

Summary (CBA/J×C57BL/6J) F2 hybrid mice (1-4) containing human variableand constant region antibody transgenes for both heavy and light chainswere immunized with recombinant human TNFα. One fusion, named GenTNV,yielded eight totally human IgG1κ monoclonal antibodies that bind toimmobilized recombinant human TNFα. Shortly after identification, theeight cell lines were transferred to Molecular Biology for furthercharacterization. As these Mabs are totally human in sequence, they areexpected to be less immunogenic than cA2 (Remicade) in humans.

ABBREVIATIONS

BSA—bovine serum albumin; CO₂—carbon dioxide; DMSO—dimethyl sulfoxide;EIA—enzyme immunoassay; FBS—fetal bovine serum; H₂O₂-hydrogen peroxide;HC—heavy chain; HRP—horseradish peroxidase; ID—interadermal;Ig—immunoglobulin; TNF—tissue necrosis factor alpha; IP—intraperitoneal;IV—intravenous; Mab—monoclonal antibody; OD—optical density;OPD—o-Phenylenediamine dihydrochloride; PEG—polyethylene glycol;PSA—penicillin, streptomycin, amphotericin; RT—room temperature;SQ—subcutaneous; TNFα—tumor necrosis factor alpha; v/v—volume pervolume; w/v—weight per volume.

Introduction.

Transgenic mice that contain human heavy and light chain immunoglobulingenes were utilized to generate totally human monoclonal antibodies thatare specific to recombinant human TNFα. It is hoped that these uniqueantibodies can be used, as cA2 (Remicade) is used to therapeuticallyinhibit the inflammatory processes involved in TNFα-mediated diseasewith the benefit of increased serum half-life and decreased side effectsrelating to immunogenicity.

As defined herein, the term “half-life” indicates that the plasmaconcentration of a drug (e.g., a therapeutic anti-TNFα antibody) ishalved after one elimination half-life. Therefore, in each succeedinghalf-life, less drug is eliminated. After one half-life the amount ofdrug remaining in the body is 50% after two half-lives 25%, etc. Thehalf-life of a drug depends on its clearance and volume of distribution.The elimination half-life is considered to be independent of the amountof drug in the body.

Materials and Methods.

Animals.

Transgenic mice that express human immunoglobulins, but not mouse IgM orIgκ, have been developed by GenPharm International. These mice containfunctional human antibody transgenes that undergo V(D)J joining,heavy-chain class switching and somatic mutation to generate arepertoire of antigen-specific human immunoglobulins (1). The lightchain transgenes are derived in part from a yeast artificial chromosomeclone that includes nearly half of the germline human Vκ locus. Inaddition to several VH genes, the heavy-chain (HC) transgene encodesboth human μ and human γ1 (2) and/or γ3 constant regions. A mousederived from the HCo12/KCo5 genotypic lineage was used in theimmunization and fusion process to generate the monoclonal antibodiesdescribed here.

Purification of Human TNFα.

Human TNFα was purified from tissue culture supernatant from C237A cellsby affinity chromatography using a column packed with the TNFαreceptor-Fc fusion protein (p55-sf2) (5) coupled to Sepharose 4B(Pharmacia). The cell supernatant was mixed with one-ninth its volume of10× Dulbecco's PBS (D-PBS) and passed through the column at 4° C. at 4mL/min. The column was then washed with PBS and the TNFα was eluted with0.1 M sodium citrate, pH 3.5 and neutralized with 2 M Tris-HCl pH 8.5.The purified TNFα was buffer exchanged into 10 mM Tris, 0.12 M sodiumchloride pH 7.5 and filtered through a 0.2 μm syringe filter.

Immunizations. A female GenPharm mouse, approximately 16 weeks old, wasimmunized IP (200 μL) and ID (100 μL at the base of the tail) with atotal of 100 μg of TNFα (lot JG102298 or JG102098) emulsified with anequal volume of Titermax adjuvant on days 0, 12 and 28. The mouse wasbled on days 21 and 35 by retro-orbital puncture without anti-coagulant.The blood was allowed to clot at RT for one hour and the serum wascollected and titered using TNFα solid phase EIA assay. The fusion,named GenTNV, was performed after the mouse was allowed to rest forseven weeks following injection on day 28. The mouse, with a specifichuman IgG titer of 1:160 against TNFα, was then given a final IV boosterinjection of 50 μg TNFα diluted in 100 μL physiological saline. Threedays later, the mouse was euthanized by cervical dislocation and thespleen was removed aseptically and immersed in 10 mL of coldphosphate-buffered saline (PBS) containing 100 U/mL penicillin, 100μg/mL streptomycin, and 0.25 μg/mL amphotericin B (PSA). The splenocyteswere harvested by sterilely perfusing the spleen with PSA-PBS. The cellswere washed once in cold PSA-PBS, counted using a Coulter counter andresuspended in RPMI 1640 media containing 25 mM Hepes.

Cell Lines. The non-secreting mouse myeloma fusion partner, 653 wasreceived into Cell Biology Services (CBS) group on May 14, 1997 fromCentocor's Product Development group. The cell line was expanded in RPMImedium (JRH Biosciences) supplemented with 10% (v/v) FBS (Cell CultureLabs), 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine (all from JRHBiosciences) and cryopreserved in 95% FBS and 5% DMSO (Sigma), thenstored in a vapor phase liquid nitrogen freezer in CBS. The cell bankwas sterile (Quality Control Centocor, Malvern) and free of mycoplasma(Bionique Laboratories). Cells were maintained in log phase cultureuntil fusion. They were washed in PBS, counted, and viability determined(>95%) via trypan blue dye exclusion prior to fusion.

Human TNFα was produced by a recombinant cell line, named C237A,generated in Molecular Biology at Centocor. The cell line was expandedin IMDM medium (JRH Biosciences) supplemented with 5% (v/v) FBS (CellCulture Labs), 2 mM L-glutamine (all from JRH Biosciences), and 0.5:g/mL mycophenolic acid, and cryopreserved in 95% FBS and 5% DMSO(Sigma), then stored in a vapor phase liquid nitrogen freezer in CBS(13). The cell bank was sterile (Quality Control Centocor, Malvern) andfree of mycoplasma (Bionique Laboratories).

Cell Fusion. The cell fusion was carried out using a 1:1 ratio of 653murine myeloma cells and viable murine spleen cells. Briefly, spleencells and myeloma cells were pelleted together. The pellet was slowlyresuspended over a 30 second period in 1 mL of 50% (w/v) PEG/PBSsolution (PEG molecular weight of 1,450 g/mole, Sigma) at 37° C. Thefusion was stopped by slowly adding 10.5 mL of RPMI media (no additives)(JRH) (37° C.) over 1 minute. The fused cells were centrifuged for 5minutes at 750 rpm. The cells were then resuspended in HAT medium(RPMI/HEPES medium containing 10% Fetal Bovine Serum (JRH), 1 mM sodiumpyruvate, 2 mM L-glutamine, 10 μg/mL gentamicin, 2.5% Origen culturingsupplement (Fisher), 50 μM 2-mercaptoethanol, 1% 653-conditioned RPMImedia, 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) andthen plated at 200 μL/well in five 96-well flat bottom tissue cultureplates. The plates were then placed in a humidified 37° C. incubatorcontaining 5% CO₂ and 95% air for 7-10 days.

Detection of Human IgG Anti-TNFα Antibodies in Mouse Serum. Solid phaseEIAs were used to screen mouse sera for human IgG antibodies specificfor human TNFα. Briefly, plates were coated with TNFα at 1 μg/mL in PBSovernight. After washing in 0.15 M saline containing 0.02% (v/v) Tween20, the wells were blocked with 1% (w/v) BSA in PBS, 200 μL/well for 1hour at RT. Plates were either used immediately or frozen at −20° C. forfuture use. Mouse sera were incubated in two-fold serial dilutions onthe human TNFα-coated plates at 50 μL/well at RT for 1 hour. The plateswere washed and then probed with 50 μL/well HRP-labeled goat anti-humanIgG, Fc specific (Accurate) diluted 1:30,000 in 1% BSA-PBS for 1 hour atRT. The plates were again washed and 100 μL/well of thecitrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodiumphosphate, 0.01% H₂O₂ and 1 mg/mL OPD) was added for 15 minutes at RT.Stop solution (4N sulfuric acid) was then added at 25 μL/well and theOD's were read at 490 nm using an automated plate spectrophotometer.

Detection of Totally Human Immunoglobulins in Hybridoma Supernatants.Because the GenPharm mouse is capable of generating both mouse and humanimmunoglobulin chains, two separate EIA assays were used to testgrowth-positive hybridoma clones for the presence of both human lightchains and human heavy chains. Plates were coated as described above andundiluted hybridoma supernatants were incubated on the plates for onehour at 37° C. The plates were washed and probed with eitherHRP-conjugated goat anti-human kappa (Southern Biotech) antibody diluted1:10,000 in 1% BSA-HBSS or HRP-conjugated goat anti-human IgG Fcspecific antibody diluted to 1:30,000 in 1% BSA-HBSS for one hour at 37°C. The plates were then incubated with substrate solution as describedabove. Hybridoma clones that did not give a positive signal in both theanti-human kappa and anti-human IgG Fc EIA formats were discarded.

Isotyping. Isotype determination of the antibodies was accomplishedusing an EIA in a format similar to that used to screen the mouse immunesera for specific titers. EIA plates were coated with goat anti-humanIgG (H+L) at 10: g/mL in sodium carbonate buffer overnight at 4EC andblocked as described above. Neat supernatants from 24 well cultures wereincubated on the plate for one hour at RT. The plate was washed andprobed with HRP-labeled goat anti-human IgG₁, IgG₂, IgG₃ or IgG₄(Binding Site) diluted at 1:4000 in 1% BSA-PBS for one hour at RT. Theplate was again washed and incubated with substrate solution asdescribed above.

Results and Discussion. Generation of Totally Human Anti-Human TNFαMonoclonal Antibodies. One fusion, named GenTNV, was performed from aGenPharm mouse immunized with recombinant human TNFα protein. From thisfusion, 196 growth-positive hybrids were screened. Eight hybridoma celllines were identified that secreted totally human IgG antibodiesreactive with human TNFα. These eight cell lines each secretedimmunoglobulins of the human IgG1κ isotype and all were subcloned twiceby limiting dilution to obtain stable cell lines (>90% homogeneous).Cell line names and respective C code designations are listed inTable 1. Each of the cell lines was frozen in 12-vial research cellbanks stored in liquid nitrogen.

Parental cells collected from wells of a 24-well culture dish for eachof the eight cell lines were handed over to Molecular Biology group onFeb. 18, 1999 for transfection and further characterization.

TABLE 1 GenTNV Cell Line Designations C Code Name DesignationGenTNV14.17.12 C414A GenTNV15.28.11 C415A GenTNV32.2.16 C416AGenTNV86.14.34 C417A GenTNV118.3.36 C418A GenTNV122.23.2 C419AGenTNV148.26.12 C420A GenTNV196.9.1 C421A

Conclusion.

The GenTNV fusion was performed utilizing splenocytes from a hybridmouse containing human variable and constant region antibody transgenesthat was immunized with recombinant human TNFα prepared at Centocor.Eight totally human, TNFα-reactive IgG monoclonal antibodies of theIgG1κ isotype were generated. Parental cell lines were transferred toMolecular Biology group for further characterization and development.One of these new human antibodies may prove useful in anti-inflammatorywith the potential benefit of decreased immunogenicity and allergic-typecomplications as compared with Remicade.

REFERENCES

-   Taylor, et al., International Immunology 6:579-591 (1993).-   Lonberg, et al., Nature 368:856-859 (1994).-   Neuberger, M. Nature Biotechnology 14:826 (1996).-   Fishwild, et al., Nature Biotechnology 14:845-851 (1996).-   Scallon, et al., Cytokine 7:759-770 (1995).

Example 4: Cloning and Preparation of Cell Lines Expressing HumanAnti-TNFα Antibody

Summary. A panel of eight human monoclonal antibodies (mAbs) with a TNVdesignation were found to bind immobilized human TNFα with apparentlyhigh avidity. Seven of the eight mAbs were shown to efficiently blockhuTNFα binding to a recombinant TNF receptor. Sequence analysis of theDNA encoding the seven mAbs confirmed that all the mAbs had human Vregions. The DNA sequences also revealed that three pairs of the mAbswere identical to each other, such that the original panel of eight mAbscontained only four distinct mAbs, represented by TNV14, TNV15, TNV148,and TNV196. Based on analyses of the deduced amino acid sequences of themAbs and results of in vitro TNFα neutralization data, mAb TNV148 andTNV14 were selected for further study.

Because the proline residue at position 75 (framework 3) in the TNV148heavy chain was not found at that position in other human antibodies ofthe same subgroup during a database search, site-directed DNAmutagenesis was performed to encode a serine residue at that position inorder to have it conform to known germline framework e sequences. Theserine modified mAb was designated TNV148B. PCR-amplified DNA encodingthe heavy and light chain variable regions of TNV148B and TNV14 wascloned into newly prepared expression vectors that were based on therecently cloned heavy and light chain genes of another human mAb(12B75), disclosed in U.S. patent application No. 60/236,827, filed Oct.7, 2000, entitled IL-12 Antibodies, Compositions, Methods and Uses,published as WO 02/12500which is entirely incorporated herein byreference.

P3X63Ag8.653 (653) cells or Sp2/0-Ag14 (Sp2/0) mouse myeloma cells weretransfected with the respective heavy and light chain expressionplasmids and screened through two rounds of subcloning for cell linesproducing high levels of recombinant TNV148B and TNV14 (rTNV148B andrTNV14) mAbs. Evaluations of growth curves and stability of mAbproduction over time indicated that 653-transfectant clones C466D andC466C stably produced approximately 125: g/ml of rTNV148B mAb in spentcultures whereas Sp2/0 transfectant 1.73-12-122 (C467A) stably producedapproximately 25: g/ml of rTNV148B mAb in spent cultures. Similaranalyses indicated that Sp2/0-transfectant clone C476A produced 18: g/mlof rTNV14 in spent cultures.

Introduction. A panel of eight mAbs derived from human TNFα-immunizedGenPharm/Medarex mice (HCo12/KCo5 genotype) were previously shown tobind human TNFα and to have a totally human IgG1, kappa isotype. Asimple binding assay was used to determine whether the exemplary mAbs ofthe invention were likely to have TNFα-neutralizing activity byevaluating their ability to block TNFα from binding to recombinant TNFreceptor. Based on those results, DNA sequence results, and in vitrocharacterizations of several of the mAbs, TNV148 was selected as the mAbto be further characterized.

DNA sequences encoding the TNV148 mAb were cloned, modified to fit intogene expression vectors that encode suitable constant regions,introduced into the well-characterized 653 and Sp2/0 mouse myelomacells, and resulting transfected cell lines screened until subcloneswere identified that produced 40-fold more mAb than the originalhybridoma cell line.

Materials and Methods.

Reagents and Cells.

TRIZOL reagent was purchased from Gibco BRL.

Proteinase K was obtained from Sigma Chemical Company. ReverseTranscriptase was obtained from Life Sciences, Inc. Taq DNA Polymerasewas obtained from either Perkin Elmer Cetus or Gibco BRL. Restrictionenzymes were purchased from New England Biolabs. QIAquick PCRPurification Kit was from Qiagen. A QuikChange Site-Directed MutagenesisKit was purchased from Stratagene. Wizard plasmid miniprep kits andRNasin were from Promega. Optiplates were obtained from Packard.¹²⁵Iodine was purchased from Amersham. Custom oligonucleotides werepurchased from Keystone/Biosource International. The names,identification numbers, and sequences of the oligonucleotides used inthis work are shown in Table 2.

TABLE 2 Oligonucleotides used to clone, engineer, or sequence the TNV mAb genes. Name I.D. Sequence HG1-4b 1193′-TTGGTCCAGTCGGACTGG-5′(SEQ ID NO: 10) HG1-5b 3543′-CACCTGCACTCGGTGCTT-5′(SEQ ID NO: 11) HG1hg 3603′-CACTGTTTTGAGTGTGTACGGGCTTAAGTT-5′ (SEQ ID NO: 12) HG1-6 353′-GCCGCACGTGTGGAAGGG-5′ (SEQ ID NO: 13) HCK1-3E 1173′-AGTCAAGGTCGGACTGGCTTAAGTT-5′ (SEQ ID NO: 14) HuK-3′Hd 2083′-GTTGTCCCCTCTCACAATCTTCGAATTT-5′ (SEQ ID NO: 15) HVKRNAseq   343′-GGCGGTAGACTACTCGTC-5′ (SEQ ID NO: 16)BsiWI M D W T W S I (SEQ ID NO: 17) 5′14s 3665-TTTCGTACGCCACCATGGACTGGACCTGGAGCATC-3′ (SEQ ID NO: 18) 5′46s 3675′-TTTCGTACGCCACCATGGGGTTTGGGCTGAGCTG-3′ (SEQ ID NO: 19) 5′47s 3685′-TTTCGTACGCCACCATGGAGTTTGGGCTGAGCATG-3′ (SEQ ID NO: 20) 563s 3695′-TTTCGTACGCCACCATGAAACACCTGTGG1TCTTC-3′ (SEQ ID NO: 21) 573s 3705′-TTTCGTACGCCACCATGGGGTCAACCGCCATCCTC-3′ (SEQ ID NO: 22)T V T V S S BstBI (SEQ ID NO: 23) HuH-J6 3883′GTGCCAGTGGCAGAGGAGTCCATTCAAGCTTAAGTT-5′ (SEQ ID NO: 24)SalI M D M R V (SEQ ID NO: 25) LK7s 3625′-TTTGTCGACACCATGGACATGAGGGTCC(TC)C-3′ (SEQ ID NO: 26) LVgs 3635′-TTTGTCGACACCATGGAAGCCCCAGCTC-3′ (SEQ ID NO: 27)T K V D I K (SEQ ID NO: 28) Afl2 HuL-J3 3803′CTGGTTTCACCTATAGTTTG/CATTCAGAATTCGGCGCCTTT (SEQ ID NO: 29) V148-QC1399 5′-CATCTCCAGAGACAATtCCAAGAACACGCTGTATC-3′ (SEQ ID NO: 30) V148-QC2400 3′-GTAGAGGTCTCTGTTAaGGTTCTTGTGCGACATAG-5′ (SEQ ID NO: 31)

The amino acids encoded by oligonucleotide 5′14s and HuH-J6 are shownabove the sequence. The ‘M’ amino acid residue represents thetranslation start codon. The underlined sequences in oligonucleotides5′14s and HuH-J6 mark the BsiWI and BstBI restriction sites,respectively. The slash in HuH-J6 corresponds to the exon/intronboundary. Note that oligonucleotides whose sequence corresponds to theminus strand are written in a 3′-5′ orientation.

A single frozen vial of 653 mouse myeloma cells was obtained. The vialwas thawed that day and expanded in T flasks in IMDM, 5% FBS, 2 mMglutamine (media). These cells were maintained in continuous cultureuntil they were transfected 2 to 3 weeks later with the anti-TNF DNAdescribed here. Some of the cultures were harvested 5 days after thethaw date, pelleted by centrifugation, and resuspended in 95% FBS, 5%DMSO, aliquoted into 30 vials, frozen, and stored for future use.Similarly, a single frozen vial of Sp2/0 mouse myeloma cells wasobtained. The vial was thawed, a new freeze-down prepared as describedabove, and the frozen vials stored in CBC freezer boxes AA and AB. Thesecells were thawed and used for all Sp2/0 transfections described here.

Assay for Inhibition of TNF Binding to Receptor. Hybridoma cellsupernatants containing the TNV mAbs were used to assay for the abilityof the mAbs to block binding of ¹²⁵I-labeled TNFα to the recombinant TNFreceptor fusion protein, p55-sf2 (Scallon et al. (1995) Cytokine7:759-770). 50:1 of p55-sf2 at 0.5: g/ml in PBS was added to Optiplatesto coat the wells during a one-hour incubation at 37° C. Serialdilutions of the eight TNV cell supernatants were prepared in 96-wellround-bottom plates using PBS/0.1% BSA as diluent. Cell supernatantcontaining anti-IL-18 mAb was included as a negative control and thesame anti-IL-18 supernatant spiked with cA2 (anti-TNF chimeric antibody,Remicade, U.S. Pat. No. 5,770,198, entirely incorporated herein byreference) was included as a positive control. ¹²⁵I-labeled TNFα(58:Ci/:g, D. Shealy) was added to 100:1 of cell supernatants to have afinal TNFα concentration of 5 ng/ml. The mixture was preincubated forone hour at RT. The coated Optiplates were washed to remove unboundp55-sf2 and 50:1 of the ¹²⁵I-TNFα/cell supernatant mixture wastransferred to the Optiplates. After 2 hrs at RT, Optiplates were washedthree times with PBS-Tween. 100:1 of Microscint-20 was added and the cpmbound determined using the TopCount gamma counter.

Amplification of V Genes and DNA Sequence Analysis. Hybridoma cells werewashed once in PBS before addition of TRIZOL reagent for RNApreparation. Between 7×10⁶ and 1.7×10⁷ cells were resuspended in 1 mlTRIZOL. Tubes were shaken vigorously after addition of 200 μl ofchloroform. Samples were centrifuged at 4° C. for 10 minutes. Theaqueous phase was transferred to a fresh microfuge tube and an equalvolume of isopropanol was added. Tubes were shaken vigorously andallowed to incubate at room temperature for 10 minutes. Samples werethen centrifuged at 4° C. for 10 minutes. The pellets were washed oncewith 1 ml of 70% ethanol and dried briefly in a vacuum dryer. The RNApellets were resuspended with 40 μl of DEPC-treated water. The qualityof the RNA preparations was determined by fractionating 0.5 μl in a 1%agarose gel. The RNA was stored in a −80° C. freezer until used.

To prepare heavy and light chain cDNAs, mixtures were prepared thatincluded 3 μl of RNA and 1 μg of either oligonucleotide 119 (heavychain) or oligonucleotide 117 (light chain) (see Table 1) in a volume of11.5 μl. The mixture was incubated at 70° C. for 10 minutes in a waterbath and then chilled on ice for 10 minutes. A separate mixture wasprepared that was made up of 2.5 μl of 10× reverse transcriptase buffer,10 μl of 2.5 mM dNTPs, 1 μl of reverse transcriptase (20 units), and 0.4μl of ribonuclease inhibitor RNasin (1 unit). 13.5 μl of this mixturewas added to the 11.5 μl of the chilled RNA/oligonucleotide mixture andthe reaction incubated for 40 minutes at 42° C. The cDNA synthesisreaction was then stored in a −20° C. freezer until used.

The unpurified heavy and light chain cDNAs were used as templates toPCR-amplify the variable region coding sequences. Five oligonucleotidepairs (366/354, 367/354, 368/354, 369/354, and 370/354, Table 1) weresimultaneously tested for their ability to prime amplification of theheavy chain DNA. Two oligonucleotide pairs (362/208 and 363/208) weresimultaneously tested for their ability to prime amplification of thelight chain DNA. PCR reactions were carried out using 2 units ofPLATINUM™ high fidelity (HIFI) Taq DNA polymerase in a total volume of50 μl. Each reaction included 2 μl of a cDNA reaction, 10 pmoles of eacholigonucleotide, 0.2 mM dNTPs, 5 μl of 10×HIFI Buffer, and 2 mMmagnesium sulfate. The thermal cycler program was 95° C. for 5 minutesfollowed by 30 cycles of (94° C. for 30 seconds, 62° C. for 30 seconds,68° C. for 1.5 minutes). There was then a final incubation at 68° C. for10 minutes.

To prepare the PCR products for direct DNA sequencing, they werepurified using the QIAquick™ PCR Purification Kit according to themanufacturer's protocol. The DNA was eluted from the spin column using50 μl of sterile water and then dried down to a volume of 10 μl using avacuum dryer. DNA sequencing reactions were then set up with 1 μl ofpurified PCR product, 10 μM oligonucleotide primer, 4 μl BigDyeTerminator™ ready reaction mix, and 14 μl sterile water for a totalvolume of 20 μl. Heavy chain PCR products made with oligonucleotide pair367/354 were sequenced with oligonucleotide primers 159 and 360. Lightchain PCR products made with oligonucleotide pair 363/208 were sequencedwith oligonucleotides 34 and 163. The thermal cycler program forsequencing was 25 cycles of (96° C. for 30 seconds, 50° C. for 15seconds, 60° C. for 4 minutes) followed by overnight at 4° C. Thereaction products were fractionated through a polyacrylamide gel anddetected using an ABI 377 DNA Sequencer.

Site-directed Mutagenesis to Change an Amino Acid. A single nucleotidein the TNV148 heavy chain variable region DNA sequence was changed inorder to replace Pro⁷⁵ with a Serine residue in the TNV148 mAb.Complimentary oligonucleotides, 399 and 400 (Table 1), were designed andordered to make this change using the QuikChange™ site-directedmutagenesis method as described by the manufacturer. The twooligonucleotides were first fractionated through a 15% polyacrylamidegel and the major bands purified. Mutagenesis reactions were preparedusing either 10 ng or 50 ng of TNV148 heavy chain plasmid template(p1753), 5 μl of 10× reaction buffer, 1 μl of dNTP mix, 125 ng of primer399, 125 ng of primer 400, and 1 μl of Pfu DNA Polymerase. Sterile waterwas added to bring the total volume to 50 The reaction mix was thenincubated in a thermal cycler programmed to incubate at 95° C. for 30seconds, and then cycle 14 times with sequential incubations of 95° C.for 30 seconds, 55° C. for 1 minute, 64° C. for 1 minute, and 68° C. for7 minutes, followed by 30° C. for 2 minutes (1 cycle). These reactionswere designed to incorporate the mutagenic oligonucleotides intootherwise identical, newly synthesized plasmids. To rid of the originalTNV148 plasmids, samples were incubated at 37° C. for 1 hour afteraddition of 1 μl of DpnI endonuclease, which cleaves only the originalmethylated plasmid. One μl of the reaction was then used to transformEpicurian Coli XL1-Blue supercompetent E. coli by standard heat-shockmethods and transformed bacteria identified after plating onLB-ampicillin agar plates. Plasmid minipreps were prepared using theWizard™ kits as described by the manufacturer. After elution of samplefrom the Wizard™ column, plasmid DNA was precipitated with ethanol tofurther purify the plasmid DNA and then resuspended in 20 μl of sterilewater. DNA sequence analysis was then performed to identify plasmidclones that had the desired base change and to confirm that no otherbase changes were inadvertently introduced into the TNV148 codingsequence. One μl of plasmid was subjected to a cycle sequencing reactionprepared with 3 μl of BigDye mix, 1 μl of pUC19 Forward primer, and 10μl of sterile water using the same parameters described in Section 4.3.

Construction of Expression Vectors from 12B75 Genes. Several recombinantDNA steps were performed to prepare a new human IgG1 expression vectorand a new human kappa expression vector from the previously-clonedgenomic copies of the 12B75-encoding heavy and light chain genes,respectively, disclosed in U.S. patent application No. 60/236,827, filedOct. 7, 2000, entitled IL-12 Antibodies, Compositions, Methods and Uses,published as WO 02/12500, which is entirely incorporated herein byreference. The final vectors were designed to permit simple, one-stepreplacement of the existing variable region sequences with anyappropriately-designed, PCR-amplified, variable region.

To modify the 12B75 heavy chain gene in plasmid p1560, a 6.85 kbBamHI/HindIII fragment containing the promoter and variable region wastransferred from p1560 to pUC19 to make p1743. The smaller size of thisplasmid compared to p1560 enabled use of QuikChange™ mutagenesis (usingoligonucleotides BsiWI-1 and BsiWI-2) to introduce a unique BsiWIcloning site just upstream of the translation initiation site, followingthe manufacturer's protocol. The resulting plasmid was termed p1747. Tointroduce a BstBI site at the 3′ end of the variable region, a 5′oligonucleotide primer was designed with SalI and BstBI sites. Thisprimer was used with the pUC reverse primer to amplify a 2.75 kbfragment from p1747. This fragment was then cloned back into thenaturally-occurring SalI site in the 12B75 variable region and a HindIIIsite, thereby introducing the unique BstB1 site. The resultingintermediate vector, designated p1750, could accept variable regionfragments with BsiWI and BstBI ends. To prepare a version of heavy chainvector in which the constant region also derived from the 12B75 gene,the BamHI-HindIII insert in p1750 was transferred to pBR322 in order tohave an EcoRI site downstream of the HindIII site. The resultingplasmid, p1768, was then digested with HindIII and EcoRI and ligated toa 5.7 kb HindIII-EcoRI fragment from p1744, a subclone derived bycloning the large BamHI-BamHI fragment from p1560 into pBC. Theresulting plasmid, p1784, was then used as vector for the TNV Ab cDNAfragments with BsiWI and BstBI ends. Additional work was done to prepareexpression vectors, p1788 and p1798, which include the IgG1 constantregion from the 12B75 gene and differ from each other by how much of the12B75 heavy chain J-C intron they contain.

To modify the 12B75 light chain gene in plasmid p1558, a 5.7 kbSalI/AflII fragment containing the 12B75 promoter and variable regionwas transferred from p1558 into the XhoI/AflII sites of plasmid L28.This new plasmid, p1745, provided a smaller template for the mutagenesisstep. Oligonucleotides (C340salI and C340sal2) were used to introduce aunique SalI restriction site at the 5′ end of the variable region byQuikChange™ mutagenesis. The resulting intermediate vector, p1746, hadunique SalI and AflII restriction sites into which variable regionfragments could be cloned. Any variable region fragment cloned intop1746 would preferably be joined with the 3′ half of the light chaingene. To prepare a restriction fragment from the 3′ half of the 12B75light chain gene that could be used for this purpose, oligonucleotidesBAHN-1 and BAHN-2 were annealed to each other to form a double-strandedlinker containing the restriction sites BsiW1, AflII, HindII, and NotIand which contained ends that could be ligated into KpnI and SacI sites.This linker was cloned between the KpnI and SacI sites of pBC to giveplasmid p1757. A 7.1 kb fragment containing the 12B75 light chainconstant region, generated by digesting p1558 with AflII, then partiallydigesting with HindIII, was cloned between the AflII and HindII sites ofp1757 to yield p1762. This new plasmid contained unique sites for BsiWIand AflII into which the BsiWI/AflII fragment containing the promoterand variable regions could be transferred uniting the two halves of thegene.

cDNA Cloning and Assembly of Expression Plasmids. All RT-PCR reactions(see above) were treated with Klenow enzyme to further fill in the DNAends. Heavy chain PCR fragments were digested with restriction enzymesBsiWI and BstBI and then cloned between the BsiWI and BstBI sites ofplasmid L28 (L28 used because the 12B75-based intermediate vector p1750had not been prepared yet). DNA sequence analysis of the cloned insertsshowed that the resulting constructs were correct and that there were noerrors introduced during PCR amplifications. The assigned identificationnumbers for these L28 plasmid constructs (for TNV14, TNV15, TNV148,TNV148B, and TNV196) are shown in Table 3.

The BsiWI/BstBI inserts for TNV14, TNV148, and TNV148B heavy chains weretransferred from the L28 vector to the newly prepared intermediatevector, p1750. The assigned identification numbers for theseintermediate plasmids are shown in Table 2. This cloning step andsubsequent steps were not done for TNV15 and TNV196. The variableregions were then transferred into two different human IgG1 expressionvectors. Restriction enzymes EcoRI and HindIII were used to transfer thevariable regions into Centocor's previously-used IgG1 vector, p104. Theresulting expression plasmids, which encode an IgG1 of the Gm(f+)allotype, were designated p1781 (TNV14), p1782 (TNV148), and p1783(TNV148B) (see Table 2). The variable regions were also cloned upstreamof the IgG1 constant region derived from the 12B75 (GenPharm) gene.Those expression plasmids, which encode an IgG1 of the G1m(z) allotype,are also listed in Table 3.

TABLE 3 Plasmid identification numbers for various heavy and light chainplasmids. Heavy Chains Gm (f+) G1m(z) 128 vector Intermediate ExpressionExpression Mab Plasmid ID Plasmid ID Plasmid ID Plasmid ID TNV14 p1751p1777 p1781 p1786 TNV15 p1752 (ND) (ND) (ND) TNV148 p1753 p1778 p1782p1787 TNV148B p1760 p1779 p1783 p1788 TNV196 p1754 (ND) (ND) (ND) LightChains pBC vector Intermediate Expression Plasmid ID Plasmid ID PlasmidID TNV14 p1748 p1755 p1775 TNV15 p1748 p1755 p1775 TNV148 p1749 p1756p1776 TNV196 p1749 p1756 p1776

The L28 vector or pBC vector represents the initial Ab cDNA clone. Theinserts in those plasmids were transferred to an incomplete 12B75-basedvector to make the intermediate plasmids. One additional transfer stepresulted in the final expression plasmids that were either introducedinto cells after being linearized or used to purify the mAb gene insertsprior to cell transfection. (ND)=not done.

Light chain PCR products were digested with restriction enzymes SalI andSacII and then cloned between the SalI and SacII sites of plasmid pBC.The two different light chain versions, which differed by one aminoacid, were designated p1748 and p1749 (Table 2). DNA sequence analysisconfirmed that these constructs had the correct sequences. TheSalI/AflII fragments in p1748 and p1749 were then cloned between theSalI and AflII sites of intermediate vector p1746 to make p1755 andp1756, respectively. These 5′ halves of the light chain genes were thenjoined to the 3′ halves of the gene by transferring the BsiWI/AflIIfragments from p1755 and p1756 to the newly prepared construct p1762 tomake the final expression plasmids p1775 and p1776, respectively (Table2).

Cell Transfections, Screening, and Subcloning. A total of 15transfections of mouse myeloma cells were performed with the various TNVexpression plasmids (see Table 3 in the Results and Discussion section).These transfections were distinguished by whether (1) the host cellswere Sp2/0 or 653; (2) the heavy chain constant region was encoded byCentocor's previous IgG1 vector or the 12B75 heavy chain constantregion; (3) the mAb was TNV148B, TNV148, TNV14, or a new HC/LCcombination; (4) whether the DNA was linearized plasmid or purified Abgene insert; and (5) the presence or absence of the complete J-C intronsequence in the heavy chain gene. In addition, several of thetransfections were repeated to increase the likelihood that a largenumber of clones could be screened.

Sp2/0 cells and 653 cells were each transfected with a mixture of heavyand light chain DNA (8-12: g each) by electroporation under standardconditions as previously described (Knight D M et al. (1993) MolecularImmunology 30:1443-1453). For transfection numbers 1, 2, 3, and 16, theappropriate expression plasmids were linearized by digestion with arestriction enzyme prior to transfection. For example, SalI and NotIrestriction enzymes were used to linearize TNV148B heavy chain plasmidp1783 and light chain plasmid p1776, respectively. For the remainingtransfections, DNA inserts that contained only the mAb gene wereseparated from the plasmid vector by digesting heavy chain plasmids withBamHI and light chain plasmids with BsiWI and NotI. The mAb gene insertswere then purified by agarose gel electrophoresis and Qiex purificationresins. Cells transfected with purified gene inserts were simultaneouslytransfected with 3-5: g of PstI-linearized pSV2gpt plasmid (p13) as asource of selectable marker. Following electroporation, cells wereseeded in 96-well tissue culture dishes in IMDM, 15% FBS, 2 mM glutamineand incubated at 37° C. in a 5% CO₂ incubator. Two days later, an equalvolume of IMDM, 5% FBS, 2 mM glutamine, 2×MHX selection (1×MHX=0.5: g/mlmycophenolic acid, 2.5: g/ml hypoxanthine, 50: g/ml xanthine) was addedand the plates incubated for an additional 2 to 3 weeks while coloniesformed.

Cell supernatants collected from wells with colonies were assayed forhuman IgG by ELISA as described. In brief, varying dilutions of the cellsupernatants were incubated in 96-well EIA plates coated with polyclonalgoat anti-human IgG Fc fragment and then bound human IgG was detectedusing Alkaline Phosphatase-conjugated goat anti-human IgG(H+L) and theappropriate color substrates. Standard curves, which used as standardthe same purified mAb that was being measured in the cell supernatants,were included on each EIA plate to enable quantitation of the human IgGin the supernatants. Cells in those colonies that appeared to beproducing the most human IgG were passaged into 24-well plates foradditional production determinations in spent cultures and thehighest-producing parental clones were subsequently identified.

The highest-producing parental clones were subcloned to identifyhigher-producing subclones and to prepare a more homogenous cell line.96-well tissue culture plates were seeded with one cell per well or fourcells per well in of IMDM, 5% FBS, 2 mM glutamine, 1×MHX and incubatedat 37° C. in a 5% CO₂ incubator for 12 to 20 days until colonies wereapparent. Cell supernatants were collected from wells that contained onecolony per well and analyzed by ELISA as described above. Selectedcolonies were passaged to 24-well plates and the cultures allowed to gospent before identifying the highest-producing subclones by quantitatingthe human IgG levels in their supernatants. This process was repeatedwhen selected first-round subclones were subjected to a second round ofsubcloning. The best second-round subclones were selected as the celllines for development.

Characterization of Cell Subclones. The best second-round subclones werechosen and growth curves performed to evaluate mAb production levels andcell growth characteristics. T75 flasks were seeded with 1×10⁵ cells/mlin 30 ml IMDM, 5% FBS, 2 mM glutamine, and 1×MHX (or serum-free media).Aliquots of 300 μl were taken at 24 hr intervals and live cell densitydetermined. The analyses continued until the number of live cells wasless than 1×10⁵ cells/ml. The collected aliquots of cell supernatantswere assayed for the concentration of antibody present. ELISA assayswere performed using as standard rTNV148B or rTNV14 JG92399. Sampleswere incubated for 1 hour on ELISA plates coated with polyclonal goatanti-human IgG Fc and bound mAb detected with AlkalinePhosphatase-conjugated goat anti-human IgG(H+L) at a 1:1000 dilution.

A different growth curve analysis was also done for two cell lines forthe purpose of comparing growth rates in the presence of varying amountsof MHX selection. Cell lines C466A and C466B were thawed into MHX-freemedia (IMDM, 5% FBS, 2 mM glutamine) and cultured for two additionaldays. Both cell cultures were then divided into three cultures thatcontained either no MHX, 0.2×MHX, or 1×MHX (1×MHX=0.5: g/ml mycophenolicacid, 2.5: g/ml hypoxanthine, 50: g/ml xanthine). One day later, freshT75 flasks were seeded with the cultures at a starting density of 1×10⁵cells/ml and cells counted at 24 hour intervals for one week. Aliquotsfor mAb production were not collected. Doubling times were calculatedfor these samples using the formula provided in SOP PD32.025.

Additional studies were performed to evaluate stability of mAbproduction over time. Cultures were grown in 24-well plates in IMDM, 5%FBS, 2 mM glutamine, either with or without MHX selection. Cultures weresplit into fresh cultures whenever they became confluent and the olderculture was then allowed to go spent. At this time, an aliquot ofsupernatant was taken and stored at 4° C. Aliquots were taken over a55-78 day period. At the end of this period, supernatants were testedfor amount of antibody present by the anti-human IgG Fc ELISA asoutlined above.

Results and Discussion.

Inhibition of TNF binding to Recombinant Receptor.

A simple binding assay was done to determine whether the eight TNV mAbscontained in hybridoma cell supernatant were capable of blocking TNFαbinding to receptor. The concentrations of the TNV mAbs in theirrespective cell supernatants were first determined by standard ELISAanalysis for human IgG. A recombinant p55 TNF receptor/IgG fusionprotein, p55-sf2, was then coated on EIA plates and ¹²⁵I-labeled TNFαallowed to bind to the p55 receptor in the presence of varying amountsof TNV mAbs. As shown in FIG. 1, all but one (TNV122) of the eight TNVmAbs efficiently blocked TNFα binding to p55 receptor. In fact, the TNVmAbs appeared to be more effective at inhibiting TNFα binding than cA2positive control mAb that had been spiked into negative controlhybridoma supernatant. These results were interpreted as indicating thatit was highly likely that the TNV mAbs would block TNFα bioactivity incell-based assays and in vivo and therefore additional analyses werewarranted.

DNA Sequence Analysis.

Confirmation that the RNAs Encode Human mAbs.

As a first step in characterizing the seven TNV mAbs (TNV14, TNV15,TNV32, TNV86, TNV118, TNV148, and TNV196) that showed TNFα-blockingactivity in the receptor binding assay, total RNA was isolated from theseven hybridoma cell lines that produce these mAbs. Each RNA sample wasthen used to prepare human antibody heavy or light chain cDNA thatincluded the complete signal sequence, the complete variable regionsequence, and part of the constant region sequence for each mAb. ThesecDNA products were then amplified in PCR reactions and the PCR-amplifiedDNA was directly sequenced without first cloning the fragments. Theheavy chain cDNAs sequenced were >90% identical to one of the five humangermline genes present in the mice, DP-46 (FIG. 2). Similarly, the lightchain cDNAs sequenced were either 100% or 98% identical to one of thehuman germline genes present in the mice (FIG. 3). These sequenceresults confirmed that the RNA molecules that were transcribed into cDNAand sequenced encoded human antibody heavy chains and human antibodylight chains. It should be noted that, because the variable regions werePCR-amplified using oligonucleotides that map to the 5′ end of thesignal sequence coding sequence, the first few amino acids of the signalsequence may not be the actual sequence of the original TNV translationproducts, but they do represent the actual sequences of the recombinantTNV mAbs.

Unique Neutralizing mAbs.

Analyses of the cDNA sequences for the entire variable regions of bothheavy and light chains for each mAb revealed that TNV32 is identical toTNV15, TNV118 is identical to TNV14, and TNV86 is identical to TNV148.The results of the receptor binding assay were consistent with the DNAsequence analyses, i.e. both TNV86 and TNV148 were approximately 4-foldbetter than both TNV118 and TNV14 at blocking TNF binding. Subsequentwork was therefore focused on only the four unique TNV mAbs, TNV14,TNV15, TNV148, and TNV196.

Relatedness of the Four mAbs

The DNA sequence results revealed that the genes encoding the heavychains of the four TNV mAbs were all highly homologous to each other andappear to have all derived from the same germline gene, DP-46 (FIG. 2).In addition, because each of the heavy chain CDR3 sequences are sosimilar and of the same length, and because they all use the J6 exon,they apparently arose from a single VDJ gene rearrangement event thatwas then followed by somatic changes that made each mAb unique. DNAsequence analyses revealed that there were only two distinct light chaingenes among the four mAbs (FIG. 3). The light chain variable regioncoding sequences in TNV14 and TNV15 are identical to each other and to arepresentative germline sequence of the Vg/38K family of human kappachains. The TNV148 and TNV196 light chain coding sequences are identicalto each other but differ from the germline sequence at two nucleotidepositions (FIG. 3).

The deduced amino acid sequences of the four mAbs revealed therelatedness of the actual mAbs. The four mAbs contain four distinctheavy chains (FIG. 4) but only two distinct light chains (FIG. 5).Differences between the TNV mAb sequences and the germline sequenceswere mostly confined to CDR domains but three of the mAb heavy chainsalso differed from the germline sequence in the framework regions (FIG.4). Compared to the DP-46 germline-encoded Ab framework regions, TNV14was identical, TNV15 differed by one amino acid, TNV148 differed by twoamino acids, and TNV196 differed by three amino acids.

Cloning of cDNAs, Site-specific Mutagenesis, and Assembly of FinalExpression Plasmids. Cloning of cDNAs. Based on the DNA sequence of thePCR-amplified variable regions, new oligonucleotides were ordered toperform another round of PCR amplification for the purpose of adaptingthe coding sequence to be cloned into expression vectors. In the case ofthe heavy chains, the products of this second round of PCR were digestedwith restriction enzymes BsiWI and BstBI and cloned into plasmid vectorL28 (plasmid identification numbers shown in Table 2). In the case ofthe light chains, the second-round PCR products were digested with SalIand AflII and cloned into plasmid vector pBC. Individual clones werethen sequenced to confirm that their sequences were identical to theprevious sequence obtained from direct sequencing of PCR products, whichreveals the most abundant nucleotide at each position in a potentiallyheterogeneous population of molecules.

Site-specific Mutagenesis to Change TNV148.

mAbs TNV148 and TNV196 were being consistently observed to be four-foldmore potent than the next best mAb (TNV14) at neutralizing TNFαbioactivity. However, as described above, the TNV148 and TNV196 heavychain framework sequences differed from the germline frameworksequences. A comparison of the TNV148 heavy chain sequence to otherhuman antibodies indicated that numerous other human mAbs contained anIle residue at position 28 in framework 1 (counting mature sequenceonly) whereas the Pro residue at position 75 in framework 3 was anunusual amino acid at that position.

A similar comparison of the TNV196 heavy chain suggested that the threeamino acids by which it differs from the germline sequence in framework3 may be rare in human mAbs. There was a possibility that thesedifferences may render TNV148 and TNV196 immunogenic if administered tohumans. Because TNV148 had only one amino acid residue of concern andthis residue was believed to be unimportant for TNFα binding, asite-specific mutagenesis technique was used to change a singlenucleotide in the TNV148 heavy chain coding sequence (in plasmid p1753)so that a germline Ser residue would be encoded in place of the Proresidue at position 75. The resulting plasmid was termed p1760 (seeTable 2). The resulting gene and mAb were termed TNV148B to distinguishit from the original TNV148 gene and mAb (see FIG. 5).

Assembly of Final Expression Plasmids.

New antibody expression vectors were prepared that were based on the12B75 heavy chain and light chain genes previously cloned as genomicfragments. Although different TNV expression plasmids were prepared (seeTable 2), in each case the 5′ flanking sequences, promoter, and intronenhancer derived from the respective 12B75 genes. For the light chainexpression plasmids, the complete J-C intron, constant region codingsequence and 3′ flanking sequence were also derived from the 12B75 lightchain gene. For the heavy chain expression plasmids that resulted in thefinal production cell lines (p1781 and p1783, see below), the human IgG1constant region coding sequences derived from Centocor's previously-usedexpression vector (p104). Importantly, the final production cell linesreported here express a different allotype (Gm(f+)) of the TNV mAbs thanthe original, hybridoma-derived TNV mAbs (G1m(z)). This is because the12B75 heavy chain gene derived from the GenPharm mice encodes an Argresidue at the C-terminal end of the CH1 domain whereas Centocor's IgG1expression vector p104 encodes a Lys residue at that position. Otherheavy chain expression plasmids (e.g. p1786 and p1788) were prepared inwhich the J-C intron, complete constant region coding sequence and 3′flanking sequence were derived from the 12B75 heavy chain gene, but celllines transfected with those genes were not selected as the productioncell lines. Vectors were carefully designed to permit one-step cloningof future PCR-amplified V regions that would result in final expressionplasmids.

PCR-amplified variable region cDNAs were transferred from L28 or pBCvectors to intermediate-stage, 12B75-based vectors that provided thepromoter region and part of the J-C intron (see Table 2 for plasmididentification numbers). Restriction fragments that contained the 5′half of the antibody genes were then transferred from theseintermediate-stage vectors to the final expression vectors that providedthe 3′ half of the respective genes to form the final expressionplasmids (see Table 2 for plasmid identification numbers).

Cell Transfections and Subcloning. Expression plasmids were eitherlinearized by restriction digest or the antibody gene inserts in eachplasmid were purified away from the plasmid backbones. Sp2/0 and 653mouse myeloma cells were transfected with the heavy and light chain DNAby electroporation. Fifteen different transfections were done, most ofwhich were unique as defined by the Ab, specific characteristics of theAb genes, whether the genes were on linearized whole plasmids orpurified gene inserts, and the host cell line (summarized in Table 4).Cell supernatants from clones resistant to mycophenolic acid wereassayed for the presence of human IgG by ELISA and quantitated usingpurified rTNV148B as a reference standard curve.

Highest-Producing rTNV148B Cell Lines

Ten of the best-producing 653 parental lines from rTNV148B transfection2 (produced 5-10: g/ml in spent 24-well cultures) were subcloned toscreen for higher-producing cell lines and to prepare a more homogeneouscell population. Two of the subclones of the parental line 2.320,2.320-17 and 2.320-20, produced approximately 50: g/ml in spent 24-wellcultures, which was a 5-fold increase over their parental line. A secondround of subcloning of subcloned lines 2.320-17 and 2.320-20 led

The identification numbers of the heavy and light chain plasmids thatencode each mAb are shown. In the case of transfections done withpurified mAb gene inserts, plasmid p13 (pSV2gpt) was included as asource of the gpt selectable marker. The heavy chain constant regionswere encoded either by the same human IgG1 expression vector used toencode Remicade (‘old’) or by the constant regions contained within the12B75 (GenPharm/Medarex) heavy chain gene (‘new’). H1/L2 refers to a“novel” mAb made up of the TNV14 heavy chain and the TNV148 light chainPlasmids p1783 and p1801 differ only by how much of the J-C intron theirheavy chain genes contain. The transfection numbers, which define thefirst number of the generic names for cell clones, are shown on theright. The rTNV148B-producing cell lines C466 (A, B, C, D) and C467Adescribed here derived from transfection number 2 and 1, respectively.The rTNV14-producing cell line C476A derived from transfection number 3.

TABLE 4 Summary of Cell Transfections. Transfection no. Plasmids HC DNAmAb HC/LC/gpt vector format Sp2/0 653 rTNV148B 1783/1776 old linear 1 2rTNV14 1781/1775 old linear 3 — rTNV148B 1788/1776/13 new insert 4,6 5,7  rTNV14 1786/1775/13 new insert 8,10 9,11 rTNV148 1787/1776/13 newinsert 12 17 rH1/L2 1786/1776/13 new insert 13 14 rTNV148B 1801/1776 oldlinear 16

ELISA assays on spent 24-well culture supernatants indicated that thesesecond-round subclones all produced between 98 and 124: g/ml, which wasat least a 2-fold increase over the first-round subclones. These 653cell lines were assigned C code designations as shown in Table 5.

Three of the best-producing Sp2/0 parental lines from rTNV148Btransfection 1 were subcloned. Two rounds of subcloning of parental line1.73 led to the identification of a clone that produced 25: g/ml inspent 24-well cultures. This Sp2/0 cell line was designated C467A (Table5).

Highest-Producing rTNV14 Cell Lines

Three of the best-producing Sp2/0 parental lines from rTNV14transfection 3 were subcloned once. Subclone 3.27-1 was found to be thehighest-producer in spent 24-well cultures with a production of 19:g/ml. This cell line was designated C476A (Table 5).

TABLE 5 Summary of Selected Production Cell Lines and their C codes.Original Spent 24-well mAb Clone Name C code Host Cell ProductionrTNV148B 2.320-17-36 C466A 653 103:g/ml 2.320-20-111 C466B 653 102:g/ml2.320-17-4 C466C 653  98:g/ml 2.320-20-99 C466D 653 124:g/ml 1.73-12-122 C467A Sp2/0  25:g/ml rTNV14  3.27-1 C476A Sp2/0  19:g/ml

The first digit of the original clone names indicates which transfectionthe cell line derived from. All of the C-coded cell lines reported herewere derived from transfections with heavy and light chain wholeplasmids that had been linearized with restriction enzymes.

Characterization of Subcloned Cell Lines

To more carefully characterize cell line growth characteristics anddetermine mAb-production levels on a larger scale, growth curvesanalyses were performed using T75 cultures. The results showed that eachof the four C466 series of cell lines reached peak cell density between1.0×10⁶ and 1.25×10⁶ cells/ml and maximal mAb accumulation levels ofbetween 110 and 140: g/ml (FIG. 7). In contrast, the best-producingSp2/0 subclone, C467A, reached peak cell density of 2.0×10⁶ cells/ml andmaximal mAb accumulation levels of 25: g/ml (FIG. 7). A growth curveanalysis was not done on the rTNV14-producing cell line, C476A.

An additional growth curve analysis was done to compare the growth ratesin different concentrations of MHX selection. This comparison wasprompted by recent observations that C466 cells cultured in the absenceof MHX seemed to be growing faster than the same cells cultured in thenormal amount of MHX (1×). Because the cytotoxic concentrations ofcompounds such as mycophenolic acid tend to be measured over orders ofmagnitude, it was considered possible that the use of a lowerconcentration of MHX might result in significantly faster cell doublingtimes without sacrificing stability of mAb production. Cell lines C466Aand C466B were cultured either in: no MHX, 0.2×MHX, or 1×MHX. Live cellcounts were taken at 24-hour intervals for 7 days. The results didreveal an MHX concentration-dependent rate of cell growth (FIG. 8). Cellline C466A showed a doubling time of 25.0 hours in 1×MHX but only 20.7hours in no MHX. Similarly, cell line C466B showed a doubling time of32.4 hours in 1×MHX but only 22.9 hours in no MHX. Importantly, thedoubling times for both cell lines in 0.2×MHX were more similar to whatwas observed in no MHX than in 1×MHX (FIG. 8). This observation raisesthe possibility than enhanced cell performance in bioreactors, for whichdoubling times are an important parameter, could be realized by usingless MHX. However, although stability test results (see below) suggestthat cell line C466D is capable of stably producing rTNV148B for atleast 60 days even with no MHX present, the stability test also showedhigher mAb production levels when the cells were cultured in thepresence of MHX compared to the absence of MHX.

To evaluate mAb production from the various cell lines over a period ofapproximately 60 days, stability tests were performed on cultures thateither contained, or did not contain, MHX selection. Not all of the celllines maintained high mAb production. After just two weeks of culture,clone C466A was producing approximately 45% less than at the beginningof the study. Production from clone C466B also appeared to dropsignificantly. However, clones C466C and C466D maintained fairly stableproduction, with C466D showing the highest absolute production levels(FIG. 9).

Conclusion

From an initial panel of eight human mAbs against human TNFα, TNV148Bwas selected as preferred based on several criteria that includedprotein sequence and TNF neutralization potency, as well as TNV14. Celllines were prepared that produce greater than 100: g/ml of rTNV148B and19: g/ml rTNV14.

Example 5: Arthritic Mice Study Using Anti-TNF Antibodies and ControlsUsing Single Bolus Injection

At approximately 4 weeks of age the Tg197 study mice were assigned,based on gender and body weight, to one of 9 treatment groups andtreated with a single intraperitoneal bolus dose of Dulbecco's PBS(D-PBS) or an anti-TNF antibody of the present invention (TNV14, TNV148or TNV196) at either 1 mg/kg or 10 mg/kg.

Results:

When the weights were analyzed as a change from pre-dose, the animalstreated with 10 mg/kg cA2 showed consistently higher weight gain thanthe D-PBS-treated animals throughout the study. This weight gain wassignificant at weeks 3-7. The animals treated with 10 mg/kg TNV148 alsoachieved significant weight gain at week 7 of the study. (See FIG. 10).

FIG. 11A-C represent the progression of disease severity based on thearthritic index. The 10 mg/kg cA2-treated group's arthritic index waslower than the D-PBS control group starting at week 3 and continuingthroughout the remainder of the study (week 7). The animals treated with1 mg/kg TNV14 and the animals treated with 1 mg/kg cA2 failed to showsignificant reduction in AI after week 3 when compared to theD-PBS-treated Group. There were no significant differences between the10 mg/kg treatment groups when each was compared to the others ofsimilar dose (10 mg/kg cA2 compared to 10 mg/kg TNV14, 148 and 196).When the 1 mg/kg treatment groups were compared, the 1 mg/kg TNV148showed a significantly lower AI than 1 mg/kg cA2 at 3, 4 and 7 weeks.The 1 mg/kg TNV148 was also significantly lower than the 1 mg/kgTNV14-treated Group at 3 and 4 weeks. Although TNV196 showed significantreduction in AI up to week 6 of the study (when compared to theD-PBS-treated Group), TNV148 was the only 1 mg/kg treatment thatremained significant at the conclusion of the study.

Example 6: Arthritic Mice Study Using Anti-TNF Antibodies and Controlsas Multiple Bolus Doses

At approximately 4 weeks of age the Tg197 study mice were assigned,based on body weight, to one of 8 treatment groups and treated with aintraperitoneal bolus dose of control article (D-PBS) or antibody(TNV14, TNV148) at 3 mg/kg (week 0). Injections were repeated in allanimals at weeks 1, 2, 3, and 4. Groups 1-6 were evaluated for testarticle efficacy. Serum samples, obtained from animals in Groups 7 and 8were evaluated for immune response induction and pharmacokineticclearance of TNV14 or TNV148 at weeks 2, 3 and 4.

Results:

No significant differences were noted when the weights were analyzed asa change from pre-dose. The animals treated with 10 mg/kg cA2 showedconsistently higher weight gain than the D-PBS-treated animalsthroughout the study. (See FIG. 12).

FIG. 13A-C represent the progression of disease severity based on thearthritic index. The 10 mg/kg cA2-treated group's arthritic index wassignificantly lower than the D-PBS control group starting at week 2 andcontinuing throughout the remainder of the study (week 5). The animalstreated with 1 mg/kg or 3 mg/kg of cA2 and the animals treated with 3mg/kg TNV14 failed to achieve any significant reduction in AI at anytime throughout the study when compared to the d-PBS control group. Theanimals treated with 3 mg/kg TNV148 showed a significant reduction whencompared to the d-PBS-treated group starting at week 3 and continuingthrough week 5. The 10 mg/kg cA2-treated animals showed a significantreduction in AI when compared to both the lower doses (1 mg/kg and 3mg/kg) of cA2 at weeks 4 and 5 of the study and was also significantlylower than the TNV14-treated animals at weeks 3-5. Although thereappeared to be no significant differences between any of the 3 mg/kgtreatment groups, the AI for the animals treated with 3 mg/kg TNV14 weresignificantly higher at some time points than the 10 mg/kg whereas theanimals treated with TNV148 were not significantly different from theanimals treated with 10 mg/kg of cA2.

Example 7: Arthritic Mice Study Using Anti-TNF Antibodies and Controlsas Single Intraperitoneal Bolus Dose

At approximately 4 weeks of age the Tg197 study mice were assigned,based on gender and body weight, to one of 6 treatment groups andtreated with a single intraperitoneal bolus dose of antibody (cA2, orTNV148) at either 3 mg/kg or 5 mg/kg. This study utilized the D-PBS and10 mg/kg cA2 control Groups.

When the weights were analyzed as a change from pre-dose, all treatmentsachieved similar weight gains. The animals treated with either 3 or 5mg/kg TNV148 or 5 mg/kg cA2 gained a significant amount of weight earlyin the study (at weeks 2 and 3). Only the animals treated with TNV148maintained significant weight gain in the later time points. Both the 3and 5 mg/kg TNV148-treated animals showed significance at 7 weeks andthe 3 mg/kg TNV148 animals were still significantly elevated at 8 weekspost injection. (See FIG. 14).

FIG. 15 represents the progression of disease severity based on thearthritic index. All treatment groups showed some protection at theearlier time points, with the 5 mg/kg cA2 and the 5 mg/kg TNV148 showingsignificant reductions in AI at weeks 1-3 and all treatment groupsshowing a significant reduction at week 2. Later in the study theanimals treated with 5 mg/kg cA2 showed some protection, withsignificant reductions at weeks 4, 6 and 7. The low dose (3 mg/kg) ofboth the cA2 and the TNV148 showed significant reductions at 6 and alltreatment groups showed significant reductions at week 7. None of thetreatment groups were able to maintain a significant reduction at theconclusion of the study (week 8). There were no significant differencesbetween any of the treatment groups (excluding the saline control group)at any time point.

Example 8: Arthritic Mice Study Using Anti-TNF Antibodies and Controlsas Single Intraperitoneal Bolus Dose Between Anti-TNF Antibody andModified Anti-TNF Antibody

To compare the efficacy of a single intraperitoneal dose of TNV148(derived from hybridoma cells) and rTNV148B (derived from transfectedcells). At approximately 4 weeks of age the Tg197 study mice wereassigned, based on gender and body weight, to one of 9 treatment groupsand treated with a single intraperitoneal bolus dose of Dulbecco=S PBS(D-PBS) or antibody (TNV148, rTNV148B) at 1 mg/kg.

When the weights were analyzed as a change from pre-dose, the animalstreated with 10 mg/kg cA2 showed a consistently higher weight gain thanthe D-PBS-treated animals throughout the study. This weight gain wassignificant at weeks 1 and weeks 3-8. The animals treated with 1 mg/kgTNV148 also achieved significant weight gain at weeks 5, 6 and 8 of thestudy. (See FIG. 16).

FIG. 17 represents the progression of disease severity based on thearthritic index. The 10 mg/kg cA2-treated group's arthritic index waslower than the D-PBS control group starting at week 4 and continuingthroughout the remainder of the study (week 8). Both of theTNV148-treated Groups and the 1 mg/kg cA2-treated Group showed asignificant reduction in AI at week 4. Although a previous study(P-099-017) showed that TNV148 was slightly more effective at reducingthe Arthritic Index following a single 1 mg/kg intraperitoneal bolus,this study showed that the AI from both versions of the TNVantibody-treated groups was slightly higher. Although (with theexception of week 6) the 1 mg/kg cA2-treated Group was not significantlyincreased when compared to the 10 mg/kg cA2 group and the TNV148-treatedGroups were significantly higher at weeks 7 and 8, there were nosignificant differences in AI between the 1 mg/kg cA2, 1 mg/kg TNV148and 1 mg/kg TNV148B at any point in the study.

Example 9: Manufacturing Processes to Produce SIMPONI® (Golimumab)Background for Golimumab

Therapies with anti-TNFα agents have been used successfully in thetreatment of inflammatory arthritides, but the early anti-TNFα agentshad limitations with respect to safety, dosing regimen, cost, and/orimmunogenicity. To address some of the limitations, a fully humananti-TNFα mAb was developed, designated SIMPONI® (golimumab). Golimumab(also known as CNTO 148 and rTNV148B) is a fully human monoclonalantibody with an Immunoglobulin G 1 (IgG1) heavy chain isotype (Glm[z]allotype) and a kappa light chain isotype. Golimumab has a heavy chain(HC) comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ IDNO:37. The molecular weight of golimumab ranges from 149,802 to 151,064Daltons.

Golimumab forms high affinity, stable complexes with both the solubleand transmembrane bioactive forms of human tumor necrosis factor alpha(TNFα) with high affinity and specificity which prevents the binding ofTNFα to its receptors and neutralizes TNFα bioactivity. No binding toother TNFα superfamily ligands was observed; in particular, golimumabdoes not bind or neutralize human lymphotoxin. TNFα is synthesizedprimarily by activated monocytes, macrophages and T cells as atransmembrane protein that self-associates to form a bioactivehomotrimer that is rapidly released from the cell surface byproteolysis. The binding of TNFα to either the p55 or p75 TNF receptorsleads to clustering of the receptor cytoplasmic domains and initiatessignaling. Tumor necrosis factor α has been identified as a key sentinelcytokine that is produced in response to various stimuli andsubsequently promotes the inflammatory response through activation ofthe caspase-dependent apoptosis pathway and the transcription factorsnuclear factor (NF)-κB and activator protein-1 (AP-1). Tumor necrosisfactor α also modulates the immune response through its role in theorganization of immune cells in germinal centers. Elevated expression ofTNFα has been linked to chronic inflammatory diseases such as rheumatoidarthritis (RA), as well as spondyloarthropathies such as psoriaticarthritis (PsA) and ankylosing spondylitis (AS). TNFα is an importantmediator of the articular inflammation and structural damage that arecharacteristic of these diseases.

Clinical Trials with Golimumab

In a global, randomized, double-blind, placebo-controlled Phase 3 studyof subcutaneously (SC) administered golimumab in subjects withAnkylosing Spondylitis (AS) (Study C0524T09), golimumab was demonstratedto be efficacious in improving the signs and symptoms, physicalfunction, and health-related quality of life (HRQOL) in subjectsaffected by Ankylosing Spondylitis (AS). Furthermore, safety analysesshowed that SC golimumab was generally well tolerated and demonstrated asafety profile similar to that observed with other anti-TNFα agents.

Given the known safety and efficacy of SC golimumab, it was anticipatedthat IV golimumab would also prove efficacious with an acceptable safetyprofile consistent with other anti-TNFα agents in rheumatologic diseasessuch as RA, PsA, and AS. Intravenous golimumab has been definitivelystudied in a Phase 3 study (CNTO148ART3001) that formed the basis ofapproval for the treatment of RA. The CNTO148ART3001 study was arandomized, double-blind, placebo-controlled, multicenter, 2-arm studyof the efficacy and safety of IV administration of golimumab 2 mg/kginfusions administered over a period of 30±10 minutes at Weeks 0, 4, andevery 8 weeks (q8w) thereafter in subjects with active RA despiteconcurrent methotrexate (MTX) therapy. Subjects with active RA despiteMTX were randomized to receive either placebo infusions or IV golimumabadministered 2 mg/kg at Weeks 0, 4, and every 8 weeks through Week 24.Starting at Week 24, all subjects were treated with IV golimumab throughWeek 100. It was demonstrated that IV golimumab provided substantialbenefits in improving RA signs and symptoms, physical function, andhealth related quality of life, as well as inhibiting the progression ofstructural damage. Golimumab administered intravenously in the treatmentof RA (CNTO148ART3001) demonstrated robust efficacy and an acceptablesafety profile with a low incidence of infusion reactions.

More recently, two Phase 3 studies were designed to evaluate theefficacy and safety of intravenous (IV) golimumab in the treatment ofsubjects with active Ankylosing Spondylitis (AS) and active PsoriatricArthritis (PsA). The IV route of administration in subjects is beingevaluated since currently available IV anti-TNFα agents have limitationswith respect to immunogenicity, infusion reactions, and have longerinfusion times (60 to 120 minutes) compared with the 30±10-minuteinfusions with IV golimumab. Patients may also prefer the maintenancedosage schedule IV golimumab rather than more frequent administrationscompared with SC agents.

Manufacturing Process Overview

Simponi (golimumab) is manufactured in a 9-stage process that includescontinuous perfusion cell culture followed by purification. An overviewof the manufacturing process is provided in FIG. 18.

As used herein, the terms “culture”, “culturing”, “cultured”, and “cellculture” refer to a cell population that is suspended in a medium underconditions suitable to survival and/or growth of the cell population. Aswill be clear from context to those of ordinary skill in the art, theseterms as used herein also refer to the combination comprising the cellpopulation and the medium in which the population is suspended. Cellculture includes, e.g., cells grown by batch, fed-batch or perfusioncell culture methods and the like. In certain embodiments, the cellculture is a mammalian cell culture.

Cell lines for use in the present invention include mammalian cell linesincluding, but not limited to, Chinese hamster vary cells (CHO cells),human embryonic kidney cells (HEK cells), baby hamster kidney cells (BHKcells), mouse myeloma cells (e.g., NS0 cells and Sp2/0 cells), and humanretinal cells (e.g., PER.C6 cells).

As used herein, the terms “chemically defined medium” or “chemicallydefined media” refer to a synthetic growth medium in which the identityand concentration of all the components are known. Chemically definedmedia do not contain bacterial, yeast, animal, or plant extracts, animalserum or plasma although they may or may not include individual plant oranimal-derived components (e.g., proteins, polypeptides, etc).Chemically defined media may contain inorganic salts such as phosphates,sulfates, and the like needed to support growth. The carbon source isdefined, and is usually a sugar such as glucose, lactose, galactose, andthe like, or other compounds such as glycerol, lactate, acetate, and thelike. While certain chemically defined media also use phosphate salts asa buffer, other buffers may be employed such as citrate,triethanolamine, and the like. As used herein, a chemically definedmedium contains no more than micromolar amounts of any metal ion.Examples of commercially available chemically defined mediums include,but are not limited to, ThermoFisher's CD Hybridoma Medium and CDHybridoma AGT™ Medium, various Dulbecco's Modified Eagle's (DME) mediums(Sigma-Aldrich Co; SAFC Biosciences, Inc), Ham's Nutrient Mixture(Sigma-Aldrich Co; SAFC Biosciences, Inc), combinations thereof, and thelike. Methods of preparing chemically defined mediums are known in theart, for example in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO2007/077217, and U.S. Patent Application Publication Nos. 2008/0009040and 2007/0212770.

The term “bioreactor” as used herein refers to any vessel useful for thegrowth of a cell culture. The bioreactor can be of any size so long asit is useful for the culturing of cells. In certain embodiments, suchcells are mammalian cells. Typically, the bioreactor will be at least 1liter and may be 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000,12,000 liters or more, or any volume in between. The internal conditionsof the bioreactor, including, but not limited to pH and temperature, areoptionally controlled during the culturing period. The bioreactor can becomposed of any material that is suitable for holding mammalian cellcultures suspended in media under the culture conditions of the presentinvention, including glass, plastic or metal. The term “productionbioreactor” as used herein refers to the final bioreactor used in theproduction of the polypeptide or glycoprotein of interest. The volume ofthe production bioreactor is typically at least 500 liters and may be1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volumein between. One of ordinary skill in the art will be aware of and willbe able to choose suitable bioreactors for use in practicing the presentinvention.

For large-scale production of golimumab expressed in Sp2/0 cells,preculture, cell expansion, and cell production are performed in Stages1 and 2. In Stage 1, preculture is initiated from a single working cellbank vial of transfected Sp2/0 cells expressing the HC and LC sequencesof golimumab and the cells are expanded in culture flasks, disposableculture bags, and either a 50-L perfusion seed bioreactor equipped withan internal spin filter or a 200-L perfusion seed bioreactor equippedwith an alternating tangential flow hollow-fiber filter (ATF) cellretention system. The cells are cultured until the cell density andvolume required for inoculation of a 500-L or a 1000-L productionbioreactor are obtained. In Stage 2, the cell culture is continuouslyperfused in a 500-L or a 1000-L production bioreactor using an ATFsystem. Cell culture permeate (harvest) is collected from the ATF systemwhile cells are returned to the bioreactor, and the culture isreplenished with fresh medium. Biomass removed from the bioreactor maybe combined with harvest withdrawn from the ATF system and then may beclarified to create a pooled harvest for further processing.

Purification of golimumab from the cell culture harvest is performed inStages 3 through 8 by a combination of affinity and ion exchangechromatography steps, and steps to inactivate or remove potential viruscontamination (solvent/detergent treatment and virus removalfiltration). In Stage 3, harvest and/or pooled harvest is clarified andpurified using Protein A affinity chromatography. The resultant directproduct capture (DPC) eluate is frozen until further processing. DPCeluates are filtered and pooled in Stage 4 following thaw, andsubsequently treated in Stage 5 with tri-n-butyl phosphate (TNBP) andpolysorbate 80 (PS 80) to inactivate any lipid-enveloped virusespotentially present.

In Stage 6, TNBP and PS 80 reagents and impurities are removed from thegolimumab product using cation exchange chromatography. The golimumabproduct is further purified using anion exchange chromatography in Stage7 to remove DNA, potentially present viruses, and impurities. In Stage8, the purified golimumab product is diluted and filtered through avirus retentive filter.

Final preparation of golimumab is performed in Stage 9. Theultrafiltration step concentrates the golimumab product, and thediafiltration step adds the formulation excipients and removes thein-process buffer salts. PS 80 is added, and the intermediate isfiltered into polycarbonate containers for frozen storage as formulatedbulk (FB) to be used for drug substance (DS) and drug product (DP).

As used herein, the terms “drug substance” (abbreviated as “DS”) and“drug product” (abbreviated as “DP”) refer to compositions for use ascommercial drugs, for example in clinical trials or as marketed drugs. ADS is an active ingredient that is intended to furnish pharmacologicalactivity or other direct effect in the diagnosis, cure, mitigation,treatment, or prevention of disease or to affect the structure or anyfunction of the human body. A DP (also referred to as a medicinalproduct, medicine, medication, or medicament) is a drug used in thediagnosis, cure, mitigation, treatment, or prevention of disease or toaffect the structure or any function of the human body. The formulatedbulk (FB) produced in the manufacturing process is the drug substance(DS). The DP is the DS that has been prepared as the medicinal productfor sale and/or administration to the patient.

Description of Cell Culture in Large-Scale Manufacturing Process withSp2/0 Cells

Stage 1 Preculture and Expansion

The first stage in the production of Simponi (golimumab) is theinitiation of preculture from a Working Cell Bank (WCB) vial oftransfected Sp2/0 cells expressing the HC and LC sequences of golimumaband subsequent expansion of the cell culture in culture flasks,disposable culture bags, and 50- or 200-L seed bioreactor. The cells arecultured until the cell density and volume required for inoculation ofthe 500- or 1000-L production bioreactor are obtained. A flow diagram ofStage 1 depicting the preculture and expansion steps with in-processcontrols and process monitoring tests are provided in FIG. 19.

Manufacturing Procedure

A cryovial from the WCB is thawed and diluted to a seeding density of0.2-0.4×10⁶ viable cells (VC)/mL with chemically defined mediumsupplemented with 6 mM L-glutamine, 0.5 mg/L mycophenolic acid, 2.5 mg/Lhypoxanthine, a n d 50 mg/L xanthine (CD-A medium). Culture viability atthaw must be 50%. The initial passage is maintained in culture flask(s)in a humidified CO₂ incubator with temperature and CO₂ controlled. Theculture is incubated for 2-3 days until a minimum cell density of0.6×10⁶ VC/mL is obtained.

Scale-up is accomplished by sequentially expanding the culture inculture flasks and disposable culture bags. Each passage is started at acell density of 0.2-0.4×10⁶ VC/mL by dilution with CD-A medium. Passagesare incubated for 2-3 days at each expansion step until a minimum celldensity of 0.6×10⁶ VC/mL is obtained. Once sufficient culture volume isachieved in a disposable culture bag at ≥0.8×10⁶ VC/mL and ≥80% cultureviability, the culture may be inoculated into the 50- or 200-L seedbioreactor.

Each preculture passage is sampled for viable cell density (VCD),culture viability, and microscopic examination. Prior to inoculation ofthe 50- or 200-L seed bioreactor, the preculture is sampled forbioburden. Preculture may be maintained for a maximum of 30 dayspost-thaw. Preculture is terminated if microbial contamination isdetected or the maximum duration is exceeded. A back-up preculture maybe retained upon inoculation of the seed bioreactor or may be startedwith a new WCB vial thaw. The back-up preculture is expanded asdescribed above and is subject to the same in-process controls andoperating parameters as the primary cultures. A back-up preculture maybe maintained and used to inoculate a 50- or 200-L seed bioreactor asneeded.

When the preculture meets inoculation criteria, the contents of thedisposable culture bag(s) are transferred to the 50- or 200-L seedbioreactor to achieve a seeding density of ≥0.3×10⁶ VC/mL. The 50- or200-L seed bioreactor is fed with CD-A culture medium and, at fullworking volume, is operated in perfusion mode. The culture is controlledfor pH, temperature, and dissolved oxygen concentration to support cellgrowth. The 50- or 200-L seed bioreactor culture is expanded until acell density of ≥2.0×10⁶ VC/mL, at 80% culture viability, is obtained.The 50- or 200-L seed bioreactor culture is sampled throughout theprocess for VCD, culture viability, and microscopic examination. Priorto inoculation of the 500- or 1000-L production bioreactor, the 50- or200-L seed bioreactor is sampled for bioburden. If the VCD of the 50- or200-L seed bioreactor reaches ≥2.0×10⁶ VC/mL and the 500- or 1000-Lproduction bioreactor is not ready for inoculation, the culture may becontinued in perfusion mode up to the maximum culture duration of 6 dayspost inoculation of the 50-L seed bioreactor and 7 days post inoculationof the 200-L seed bioreactor. The 50- or 200-L seed bioreactor operationis terminated if microbial contamination is detected or the maximumduration is exceeded.

Stage 2 Bioreactor Production

The second stage in the manufacturing process is perfusion cell culturein a 500- or 1000-L production bioreactor. Cell culture permeate(harvest) is collected from the production bioreactor while cells areretained via an alternating tangential flow (ATF) hollow fibercell-retention device, and the culture is replenished with fresh media.A flow diagram depicting the 500- or 1000-L production bioreactorprocess is provided in FIG. 20.

Manufacturing Procedure

The inoculation of the 500-L or 1000-L production bioreactor isperformed by transferring the contents of the 50- or 200-L seedbioreactor into the 500- or 1000-L production bioreactor containingchemically defined medium supplemented with 6 mM L-glutamine, 0.5 mg/Lmycophenolic acid, 2.5 mg/L hypoxanthine, and 50 mg/L xanthine (CD-Amedium). The volume transferred must be sufficient to yield a seedingdensity of ≥10⁶ viable cells (VC)/mL. The cultures are maintained at atemperature of 34.0-38.0° C., a pH of 6.80-7.40, and dissolved oxygenconcentration of 10-80%. Sampling is performed throughout the 500- or1000-L production process for viable cell density (VCD), cultureviability, bioburden, and immunoglobulin G (IgG) concentration.

After inoculation, the medium feed rate to the culture is increasedaccording to a predetermined schedule until the maximum feed rate isreached. The maximum feed rate is controlled to 0.80-1.50 reactorvolumes per day. When the full working volume of the bioreactor isreached, perfusion is initiated using the ATF system to separate cellsfrom the permeate. Permeate is continuously withdrawn through the ATFfilter, while cell culture is cycled between the ATF system and thebioreactor. The ATF permeate is collected in bioprocess containers(BPCs).

The medium feed to the bioreactor is switched from CD-A to chemicallydefined medium supplemented with 6 mM L-glutamine, 0.5 mg/L mycophenolicacid, 2.5 mg/L hypoxanthine, 50 mg/L xanthine, and 10 mM sodium acetate(CD-B medium) when the VCD reaches ≥10⁶ VC/mL, but no later than Day 15post inoculation of the 500- or 1000-L production bioreactor. The viablecell density in the bioreactor is controlled to a target of at least12.0×10⁶VC/mL by means of a variable biomass removal flow from theculture.

Biomass removed from the bioreactor may be discarded or combined withthe ATF permeate and clarified by filtration.

The ATF permeate is designated as the harvest stream.Ethylenediaminetetraacetic acid (EDTA) is added to the harvest stream toa concentration of 5-20 mM. The harvest is stored in bioprocesscontainers (BPCs) in a 2-8° C. environment for a maximum period of 21days after disconnection from the bioreactor. Each harvest BPC issampled for IgG concentration, endotoxin, and bioburden prior to directproduct capture (Stage 3).

Perfusion cell culture operation in the 500- or 1000-L productionbioreactor continues for up to 60 days post inoculation. On the finalday of the 500- or 1000-L production bioreactor operation, the cultureis sampled for mycoplasma and adventitious virus testing. The bioreactorIgG concentration is monitored and reported for information only.

Description for Small-Scale Production of Golimumab Expressed in CHOCells Generation of CHO Cells Expressing Golimumab

The CHO cell line was originally created by T. T. Puck from the ovary ofan adult Chinese hamster. CHO-K1 (ATCC® CCL-61) is a subclone of theparental CHO cell line that lacks the proline synthesis gene. CHO-K1 wasalso deposited at the European Collection of Cell Cultures, CHO-K1(ECACC 85051005). A master cell bank (MCB) of CHO-K1, 024 M, wasestablished at Celltech Biologics (now Lonza Biologics) and used foradaptation of CHO-K1 to suspension culture and serum-free medium. Theadapted cell line was named CHOK1SV. The CHOK1SV cell line was furtheradapted in protein-free medium to create a MCB of cells referred to as269-M. Cells derived from the 269-M MCB were transfected as describedbelow to create the CHO cell lines expressing golimumab.

Cell lines were generated, expanded, and maintained in a humidifiedincubator at 37° C. and 5% CO₂ using cell culture plates and shakeflasks. Routine seeding density in shake flasks was 3×10⁵ viable cellsper mL (vc/mL). All shake flask cultures were maintained at 130revolutions per minute (rpm) with a 25 mm orbit and 96-deepwell (DW,Thermo Scientific, Waltham, Mass., Cat. #278743) cultures weremaintained at 800 rpm with a 3 mm orbit.

CHO clones expressing golimumab were created using media identified asMACH-1, an in-house developed, chemically-defined medium for CHO cellculture. The basal medium for the routine passage of the CHO host cellline was MACH-1 supplemented with 6 mM L-glutamine (Invitrogen,Carlsbad, Calif., Cat. #25030-081). CHO cells transfected with theglutamine synthetase (GS) gene were grown in MACH-1+ MSX unlessotherwise noted, which is MACH-1 supplemented with 25 μM L-methioninesulfoximine (MSX, Sigma, St. Louis, Mo., Cat. #M5379-1G) to inhibitglutamine synthetase function. For bolus fed-batch shake flask andbioreactor experiments, cells were cultured in MACH-1+ F8, which isMACH-1 supplemented with 8 g/kg F8 (a supplement of proprietary growthenhancers) to further support cell growth and antibody production.Proprietary feed media were used in shake flask and bioreactorexperiments.

The DNA encoding the genes of interest were cloned into aglutamine-synthetase (GS) double gene expression plasmid (LonzaBiologics). Expression of the heavy chain (HC) and light chain (LC)genes were driven by separate human cytomegalovirus (hCMV-MIE)promoters. The GS gene selection marker, driven by the Simian Virus SV40promoter, allows for the selection of transfected cells inglutamine-free media in the presence of MSX.

Prior to each transfection, 1 aliquot of plasmid DNA, containing boththe HC and LC coding regions of golimumab, was linearized by restrictionenzyme digestion. A linearized 15 μg DNA aliquot was transfected into a1×10⁷ cell aliquot using the BTX ECM 830 Electro Cell Manipulator(Harvard Apparatus, Holliston, Mass.). Cells were electroporated 3 timesat 250 volts with 15 millisecond pulse lengths and 5 second pulseintervals in a 4 mm gap cuvette. Transfected cells were transferred toMACH-1+ L-glutamine in a shake flask and incubated for 1 day.Transfections were centrifuged, then resuspended in MACH-1+25 uM MSX forselection and transferred to shake flasks to incubate for 6 days.

Following chemical selection, cells were plated in a single cellsuspension in custom glutamine-free Methocult medium containing 2.5%(w/v) methylcellulose in a Dulbecco's Modified Eagle's Medium (DMEM)base media (Methocult, StemCell Technologies, Inc., Vancouver, BC, Cat.#03899). The working solution also contained 30% (v/v) gamma-irradiateddialyzed fetal bovine serum (dFBS.IR, Hyclone, Logan, Utah, Cat.#SH30079.03), lx GS Supplement (SAFC, St. Louis, Mo., Cat. #58672-100M),1.5 mg animal component-free Protein G Alexa Fluor 488 conjugate(Protein G, Invitrogen, Carlsbad, Calif., Cat. #C47010), 25 μM MSX,Dulbecco's Modified Eagle's Medium with F12 (DMEM/F12, Gibco/Invitrogen,Carlsbad, Calif., Cat. #21331-020), and cell suspension.

Protein G recognizes human monoclonal antibodies and binds to the IgGthat is secreted by the cells. The Protein G is conjugated to thefluorescent label Alexa Fluor 488, so that cell colonies secreting themost antibodies will show the highest levels of fluorescence. Afterincubation for 12 to 18 days, colonies with the highest fluorescencelevels were picked into 100 μL phenol red-containing MACH-1+ MSX in96-well plates using a ClonePix FL colony picking instrument (MolecularDevices, Sunnyvale, Calif.) and incubated without shaking for 5-7 days.After 5-7 days, cells from the 96-well plate were expanded by adding to50-100 μL phenol red-containing MACH-1+ MSX in a 96DW plate (ThermoScientific, Waltham, Mass., Cat. #278743) and shaken at 800 rpm with a 3mm orbit. The 96DW plates were fed and at 7 days post 96DW seeding weretitered via Octet (ForteBio, Menlo Park, Calif.). The top 10 culturescorresponding to the highest batch 96DW overgrow titers were expanded toshake flasks in MACH-1+ MSX, and frozen cell banks were created withcells suspended in in MACH-1+ MSX medium containing 10% DMSO.

Cell Culture for Small-Scale Production

As in large-scale production of golimumab expressed in Sp2/0 cells,preculture, cell expansion, and cell production are performed in Stages1 and 2 for small-scale production of golimumab expressed in ChineseHamster Ovary cells (CHO cells). In Stage 1, preculture is initiatedfrom a single cell bank vial of transfected CHO cells expressing the HCand LC sequences of golimumab and the cells are expanded in cultureflasks. The cells are cultured until the cell density and volumerequired for inoculation of a 10-L production bioreactor are obtained.In Stage 2, the cell culture is run in fed-batch mode in a 10-Lproduction bioreactor. For the duration of the 15-day bioreactor run theculture is fed as required with concentrated glucose-based and aminoacid-based feeds. At the completion of the production bioreactor runcell culture harvest is clarified to remove biomass and filtered forfurther processing.

Purification for Small-Scale Production

The purification steps for small-scale production of golimumab wereidentical to the large-scale manufacturing process, except the Stage 8virus filtration step was omitted for small-scale production. In brief,for small-scale production, purification of golimumab from the cellculture harvest is performed in Stages 3 through 7 by a combination ofaffinity and ion exchange chromatography steps and steps to inactivateor remove potential virus contamination (solvent/detergent treatment andvirus removal). In Stage 3, harvest and/or pooled harvest is clarifiedand purified using Protein A affinity chromatography. The resultantdirect product capture (DPC) eluate is frozen until further processing.DPC eluates are filtered and pooled in Stage 4 following thaw, andsubsequently treated in Stage 5 with tri-n-butyl phosphate (TNBP) andpolysorbate 80 (PS 80) to inactivate any lipid-enveloped virusespotentially present.

In Stage 6, TNBP and PS 80 reagents and impurities are removed from thegolimumab product using cation exchange chromatography. The golimumabproduct is further purified using anion exchange chromatography in Stage7 to remove DNA, potentially present viruses, and impurities. As notedabove, Stage 8 filtering through a virus retentive filter was omittedfrom the CHO derived golimumab product purification process.

Final preparation of golimumab is performed in Stage 9 (reference tolarge-scale stages). The ultrafiltration step concentrates the golimumabproduct, and the diafiltration step adds the formulation excipients andremoves the in-process buffer salts. PS 80 is added, and the bulkintermediate is filtered into polycarbonate containers for frozenstorage as formulated bulk.

Methods Methods for Determining Viable Cell Density (VCD) and %Viability

Total cells per/ml, viable cells/ml (VCD), and % viability are typicallydetermined with a Beckman Coulter Vi-CELL-XR cell viability analyzerusing manufacturer provided protocols, software and reagents.Alternatively, a CEDEX automated cell counting system has also beenused. It should also be noted, however, that other methods fordetermining VCD and % viability are well known by those skilled in theart, e.g., using a hemocytometer and trypan blue exclusion.

Bioactivity (Potency) Assay

Measurement of bioactivity (potency) of golimumab is performed with anin vitro assay based on the ability of golimumab to protect WEHI 164cells (Mouse BALB/c fibrosarcoma cells, obtained from Walter and ElizaHall Institute, Melbourne, Australia) from TNFα induced cytotoxicity.Each assay plate contains 100-4 serial dilutions of 500 ng/mL (6replicates) of Simponi test article and Simponi Reference Standard.TNF-α is then added and the plates are incubated. After neutralizationand incubation, WEHI 164 cells are added to the microtiter platefollowed by another incubation step. Afterwards, a metabolic substrate(which is an indicator of live cells) is added and the convertedsubstrate is measured spectrophotometrically.

The test article and Reference Standard neutralization curves are fitusing a 4-parameter logistic analysis. The potency is calculated bycomparing the 50% effective dose (ED₅₀) of the Simponi ReferenceStandard and the Simponi test article.

The following system suitability acceptance criteria are applied duringthe performance of the bioactivity procedure in order to yield a validresult:

Reference Standard:

-   -   Each of the neutralization curves must be an S-shape curve with        a lower plateau within 40% of the average OD value of the        Cells+TNF-α controls of the 3 assay plates, a higher plateau        within 25% of the average OD value of the Cells Only controls of        the 3 assay plates, and a linear part between the plateaus.    -   The slope of each curve must be 0.7 and 3.5.    -   The r² value for each curve must be 0.97.    -   All replicate ED₅₀ values must be 2 ng/mL and 20 ng/mL.    -   The RSD of the average ED₅₀ values (n=6) must be <20%.

TNF-α Cytotoxicity Curve:

-   -   The TNF-α cytotoxicity curve must show an S-shape curve with a        lower plateau, upper plateau and a linear part between the        plateaus.    -   The slope must be 2.0 for each fitted curve.    -   The r² values for the TNF-α cytotoxicity curves must be 0.97.    -   The OD value at a TNF-α concentration of 1.68 ng/mL should fall        between 0.1 and 0.4.

Controls:

-   -   The OD range (difference between the mean OD values of the        Cells+TNF Control and Cells Only Control) for each plate must be        0.68.    -   The average OD value (n=6) of the Cells Only controls must be        0.75 for each plate. The RSD of the Cells Only controls must be        20%.    -   The average OD value (n=6) of the Cells+TNF-α controls must be        0.50 for each plate. And the RSD of the TNF-α controls must be        20%.

Test Articles:

-   -   Each of the neutralization curves must be an S-shape curve with        a lower plateau within 40% of the average OD value of the        Cells+TNF-α controls of the 3 assay plates, a higher plateau        within 25% of the average OD value of the Cells only controls of        the 3 assay plates, and a linear part between the plateaus.    -   The slope of each curve must be 0.7 and 3.5.    -   The r² value for each curve must be 0.97.    -   The RSD of the average ED₅₀ ratio values (n=6) must be 25%.    -   The mean slope ratio between the test article and reference        standard curves is 0.8 and 1.2, which assures that the slope        values of the test article and reference standard curves are        comparable (with no more than a 20% difference).    -   The mean upper asymptote value of the test article        neutralization curves does not differ from that of reference        standard by more than 10% (difference in mean upper asymptote        values 10%).    -   The mean lower asymptote value of the test article        neutralization curves does not differ from that of reference        standard by more than 15% (difference in mean lower asymptote        values 15%).

Methods for Determining Oligosaccharide Composition OligosaccharideComposition by HPLC

The N-linked oligosaccharide composition of golimumab is determined witha normal phase anion exchange HPLC method with fluorescent detectionusing an Agilent 1100/1200 Series HPLC System with Chemstation/Chemstoresoftware. To quantitate the relative amounts of glycans, the N-linkedoligosaccharides are first cleaved from the reduced and denatured testarticle with N-glycanase (PNGase F). The released glycans are labeledusing anthranilic acid, purified by filtration using 0.45-μm nylonfilters, and analyzed by normal phase anion exchange HPLC withfluorescence detection. The HPLC chromatogram serves as a map that canbe used to identify and quantitate the relative amounts of N-linkedoligosaccharides present in the sample. Glycans are identified byco-elution with oligosaccharide standards and by retention time inaccordance with historical results from extensive characterizations. Arepresentative HPLC chromatogram for a golimumab reference standard isshown FIG. 21.

The amount of each glycan is quantitated by peak area integration andexpressed as a percentage of total glycan peak area (peak area %).Results can be reported for G0F, G1F, G2F, total neutrals and totalcharged glycans. Other neutrals are the sum of all integrated peaksbetween 17 and 35 minutes, excluding the peaks corresponding to G0F, G1Fand G2F. Total neutral glycans is the sum of G0F, G1F, G2F and the otherneutrals. Total charged glycans is the sum of all mono-sialylated glycanpeaks eluting between 42 and 55 minutes and all di-sialylated glycanpeaks eluting between 78 and 90 minutes.

A mixture of oligosaccharide standards (G0F, G2F, G2F+N-acetylneuraminicacid (NANA) and G2F+2NANA) is analyzed in parallel as a positive controlfor the labeling reaction, as standards for peak identification, and asa measure of system suitability. Reconstituted oligosaccharides fromProzyme, G0F (Cat. No. GKC-004301), G2F (Cat. No. GKC-024301), SA1F(Cat. No. GKC-124301), and SA2F (Cat. No. GKC-224301), or equivalent,are used as reference standards. A method blank negative control andpre-labeled G0F standard are also run for system suitability purposes.The following system suitability and assay (test article) acceptancecriteria are applied during the performance of the oligosaccharidemapping procedure in order to yield a valid result:

System Suitability Criteria:

-   -   Resolution (USP) between the G0F and G2F peaks in the        oligosaccharide standard must be ≥3.0.    -   Theoretical plate count (tangent method) of the G0F peak in the        oligosaccharide standards must be ≥5000.    -   The total glycan peak area for the golimumab reference standard        must be ≥1.5 times of the major glycan peak area of the        pre-labeled G0F.    -   If any reference standard glycan peak is off-scale, the        reference standard is re-injected with less injection volume    -   The retention time of G0F peak in the golimumab reference        standard must be within 0.4 min of the G0F retention time in the        oligosaccharide standards.

Assay Acceptance Criteria:

-   -   The method blank must have no detectable peaks that co-elute        with assigned oligosaccharide peaks in golimumab.    -   The total glycan peak area of each test article must be ≥1.5        times the major glycan peak area of the pre-labeled G0F        standard.    -   If any sample glycan peak is off-scale, that sample is        re-injected with less injection volume, together with        pre-labeled G0F, the oligosaccharide standards, Method Blank and        reference standard with normal volume.    -   The retention time for the G0F peak in each test article must be        within 0.4 min of the retention time for the G0F peak in the        oligosaccharide standards.    -   If the assay fails to meet any acceptance criteria, the assay is        invalidated

Oligosaccharide Composition by IRMA

The IdeS-RMA (IRMA) method allows differentiation between majorglycoforms by Reduced Mass Analysis (RMA) after the enzymatic treatmentof immunoglobulin G (IgG) with FabRICATOR®, an IgG degrading enzyme ofStreptococcus pyogenes (IdeS) available from Genovis AB (SKU:A0-FR1-050). See also, for example, U.S. Pat. No. 7,666,582. ReducedMass Analysis (RMA) involves disulfide bond reduction of antibodiesfollowed by the intact mass analysis of the heavy chain of the antibodyand its attached glycan moieties. Some antibodies show a large degree ofheterogeneity due to the presence of N-terminal modifications such aspyroglutamate formation and carboxylation. Consequently, disulfidereduction and heavy chain mass measurement results in a complex patternof deconvoluted peaks. Therefore, in some applications, proteolyticgeneration of antibody fragments is desired over generation of light andheavy chains using reduction agents such as dithiothreitol (DTT).Traditionally papain and pepsin are used to generate antibody fragmentsall of which are laborious processes. Cleavage of IgG with pepsinrequires extensive optimization and it is done at low acidic pH. Papainneeds an activator and both F(ab′)2 and Fab can be obtained depending onthe reaction conditions resulting in a heterogeneous pool of fragments.These drawbacks can be circumvented by using the novel enzyme,FabRICATOR®. The cleavage procedure is very fast, simple, andimportantly no optimization is needed. It is performed at neutral pHgenerating precise F(ab′)2 and Fc fragments. No further degradation orover-digestion is observed as is commonly associated with otherproteolytic enzymes like pepsin or papain. Importantly, as FabRICATOR®cleaves just C-terminally of the disulfide bridges in the heavy chain,no reduction step is required and an intact F(ab′)2 and two residual Fcfragments are obtained.

Definitions

-   -   H: hexose (mannose, glucose, and galactose)    -   Man5: mannose 5    -   N: N-acetylhexosamine (N-acetylglucosamine and        N-acetylgalactosamine)    -   F: fucose    -   S: sialic acid (N-acetylneuraminic acid (NANA) and        N-glycolylneuraminic acid (NGNA))    -   G0: asialo-agalacto-afucosylated biantennary oligosaccharide    -   G0F: asialo-agalacto-fucosylated biantennary oligosaccharide    -   G1: asialo-monogalactosylated-afucosylated biantennary        oligosaccharide    -   G1F: asialo-monogalactosylated-fucosylated biantennary        oligosaccharide    -   G2: asialo-digalactosylated-afucosylated biantennary        oligosaccharide    -   G2F: asialo-digalactosylated-fucosylated biantennary        oligosaccharide    -   GlcNAc: N-Acetyl-D-Glucosamine    -   Lys: Lysine    -   −Lys: Truncated heavy chain (no C-terminal Lysine residue        present)    -   +Lys: Heavy chain containing C-terminal Lysine    -   ppm: parts per million

Equipment

-   -   Thermo Scientific Q Exactive (Plus) mass spectrometer    -   Agilent 1200 HPLC system    -   Applied Biosystems POROS R2/10 2.1 mmD×100 mmL column    -   Thermo Scientific Q Exactive Tune software    -   Thermo Scientific Protein Deconvolution software    -   Analytical balance capable of weighing 0.01 mg    -   Vortex mixer, any suitable model    -   Water bath or heating block, any suitable model    -   Calibrated Thermometer—10 to 110° C., any suitable model    -   Calibrated Pipettes    -   Microcentrifuge, any suitable model

Procedure IdeS Digestion of Samples

-   -   samples (equal to 50 μg IgG).    -   add 1 μl (50 units) of IdeS enzyme to 50 μg of IgG, vortex        briefly, spin down, and incubate at 37° C. for 30 minutes (stock        enzyme @ 5000 units per 100 μl. 1 unit of enzyme fully digests 1        μg of IgG in 30 minutes at 37° C.)    -   spin down samples and transfer to LC-MS vials, and load sample        vials into Agilent 1200 autosampler

LC-MS Method Solution Preparation

-   -   Mobile phase A (0.1% Formic Acid (FA) in ultrapure water)—Add        999 mL of ultrapure water to a 1L HPLC Mobile phase bottle, add        1 mL FA and stir. This solution can be stored at RT for 2        months.    -   Mobile phase B (0.1% FA, 99.9% acetonitrile)—Add 999 mL of        acetonitrile to a 1L HPLC Mobile phase bottle, add 1 mL FA and        stir. This solution can be stored at RT for 2 months.

LC Method

-   -   Column: Applied Biosystems POROS R2/10 2.1 mmD×100 mmL    -   Column temperature: 60° C.    -   Auto sampler temperature: 4° C.    -   Flow rate: 300 μL/min    -   Injection volume: 5 μL    -   Mobile phase A: 0.1% FA in ultrapure water    -   Mobile phase B: 0.1% FA in acetonitrile

TABLE 6 LC Gradient Table Time (mm) % Mobile phase B 0.0 10 6.0 30 11.942 12.0 95 15.9 95 16.0 10 21.0 10

MS Method

Scan Parameters:

-   -   Scan type: Full MS    -   Scan range: 700 to 3500 m/z    -   Fragmentation: In-source CID 35.0 eV    -   Resolution: 17500    -   Polarity: Positive    -   Lock masses: On, m/z 445.12002    -   AGC target: 3e6    -   Maximum injection time: 250

HESI Source:

-   -   Sheath gas flow rate: 32    -   Aux gas flow rate: 7    -   Sweep gas flow rate: 0    -   Spray voltage (IWO: 4.20    -   Capillary temp. (° C.): 280    -   S-lens RF level: 55.0    -   Heater temp. (° C.): 80

Data Analysis

The relative content of each detected glycan species is recorded basedon analysis of deconvoluted mass spectra. FIG. 22 shows a representativedeconvoluted mass spectrum for IRMA analysis of golimumab produced inSp2/0 cells. The major structures determined by IRMA analysis include,e.g., Man 5 (H5N2), G0 (H3N4), G0F (H3N4F1), G1F-GlcNAc (H4N3F1), H5N3G1 (H4N4), H5N3F1, G1F (H4N4F1), G2 (H5N4), G2F (H5N4F1), G1FS(H4N4F1S1), H6N4F1, G2FS (H5N4F1S1), H7N4F1, H6N4F1S1, G2FS2 (H5N4F1S2).The percentage of each of these structures is monitored. The measuredpeak intensity represents the percentage of each structure afternormalization (% of Total Assigned). Glycans of which the observed massis outside the 100 ppm mass deviation threshold are not included in thecalculations, e.g., *G1F-GlcNAc−Lys, *H5N3−Lys, *G1−Lys, *H5N3F1−Lys,and *G2−Lys. As noted, these are indicated with an asterisk (“*”). Also,Man5−Lys is not always detected in the spectra since it has a very lowintensity, nevertheless it is considered and included into thecalculations when present. The percentage of a glycan is calculated asdetected on both isoforms of the Fc fragment with and without terminalLysine, e.g., percentage G0F is (% G0F−Lys+% G0F+Lys). Structuresdetected on only one of the heavy chain isoforms are indicated with adouble asterisk (“**”), e.g., **G1F-G1cNAc+Lys, **H5N3+ Lys, **G1+ Lys,**H5N3F1+ Lys, **G2+ Lys, **G2FS−Lys, **H6N4F1S1−Lys, **G2FS2−Lys,**H6N4F1−Lys, **H7N4F1−Lys. Most of these structures are low abundantand cannot be resolved from adjacent peaks with higher intensities orare below the detection capabilities of the method.

*Note:

Differences between the HPLC and IRMA methods (e.g., see Table 7 below)may result from co-elution of species in HPLC and possiblyunderestimation of some sialylated species by IRMA because some of theintensities are very close to the detection capabilities of the IRMAmethod.

TABLE 7 Glycan abundance comparison for IRMA and HPLC for arepresentative golimumab sample produced in Sp2/0 cells Glycan GroupIRMA % HPLC % G0F 28.7 32.5 G1F 31.7 36.4 G2F 10.5 9.9 Other neutraloligosaccharides 15.2 6.8 Total neutral oligosaccharides 86.1 85.7Monosialylated 12.6 13.8 Disialylated 1.3 0.6 Total chargedoligosaccharides 13.9 14.3

Capillary Isoelectric Focusing

Capillary isoelectric focusing (cIEF) separates proteins on the basis ofoverall charge or isoelectric point (pI). The method is used to monitorthe distribution of charge-based isoforms in golimumab. Unlike thegel-based IEF procedures, cIEF provides a quantitative measure of thecharged species present. In addition, cIEF shows increased resolution,sensitivity, and reproducibility compared to the gel-based method. ThecIEF procedure separates 4 to 6 charge-based isoforms of Simponi(golimumab) with nearly baseline resolution, while IEF gel analysisseparates only 4 to 5 species with partial resolution. A representativecIEF electropherogram of golimumab expressed in Sp2/0 cells is shown inFIG. 23, with the four major peaks labeled as C, 1, 2, and 3 and minorpeak labeled B. A graphic representing the general relationship betweencIEF peaks and decreasing negative charge/degree of sialylation is alsoshown.

The cIEF assay is performed on a commercially available imaging cIEFanalyzer equipped with an autosampler able to maintain sampletemperature ≤10.5° C. in an ambient environment of <30° C., such as theAlcott autosampler (GP Instruments, Inc.). The analysis employs an innerwall-coated silica capillary without an outer wall polyimide coating toallow for whole column detection. In addition, an anolyte solution ofdilute phosphoric acid and methylcellulose, a catholyte solution ofsodium hydroxide and methylcellulose, and a defined mixture of broadrange (pH 3-10) and narrow range (pH 8-10.5) ampholytes are used. Theassay employs a pre-treatment of both test articles and ReferenceStandard (RS) with carboxypeptidase B (CPB) which removes the heavychain C-terminal lysine and eliminates ambiguities introduced by thepresence of multiple C-terminal variants.

Before each analysis, the autosampler temperature set-point is set to 4°C. and the autosampler is pre-cooled for at least 30 minutes and theambient room temperature of the lab is maintained ≤30° C. Thepre-treated test article and RS, sample vials, vial inserts, thereagents used in the assay including purified water, the parent solutioncontaining N,N,N′,N′-Tetramethylethylenediamine (TEMED) (which optimizesfocusing within the capillary), ampholytes, pI 7.6 and 9.5 markers forinternal standards and methylcellulose (MC) are kept on ice for at least30 minutes before starting sample preparation. The samples are preparedon ice and the time of addition of the parent solution is recorded andexposure to TEMED is controlled. The assay must be completed within 180minutes after this addition. System suitability controls are injectedonce, and test articles and RS are injected twice following the sequencetable below (Table 8):

TABLE 8 Sample Running Sequence Sample Vial Number of Sample NamePosition Injections System Suitability 1 1 Blank 2 1 CPB Control 3 1 CPBTreated RS 4 2 CPB Treated Sample 1 5 2 CPB Treated RS 6 2

After the samples are injected into the capillary by a syringe pump, anelectric field (3 kV) is applied across the capillary for 8 min, forminga pH gradient, and charge-based isoforms of golimumab are separatedaccording to their isoelectric point (pI). The protein isoforms in thecapillary are detected by imaging the entire capillary at 280 nm, andthe data are presented in the form of an electropherogram as a functionof pI value vs A280. Values for pI are assigned by comparison to theinternal pI standards (pI 7.6 and 9.5) using the instrument software,and peak areas are determined from the electropherogram using standarddata acquisition software. The average pI and average peak areapercentage from duplicate injections of all peaks ≥LOQ, the ΔpI valuecompared to Reference Standard, and the sum of percent area of peaks C,1, 2, and 3 are reported.

Characterization of Golimumab cIEF Isoforms

The reference cIEF profile of golimumab produced on Sp2/0 cells containsfour major peaks labeled as C, 1, 2, and 3 and minor peak B as shown inthe representative electropherogram (FIG. 23). For golimumab, the majorsource of variability in the cIEF profile is the deamidation andisomerization of heavy chain asparagine 43 (HC Asn43). Basic Peak 3 incIEF represents non-deamidated HC Asn43 as well as deamidated heavychain isoaspartic acid 43 (HC isoAsp43), while the more acidic peaksrepresent deamidated HC Asp43 and degree of sialylation. The predictedidentities of the different cIEF peaks are listed in Table 9. The cIEFassay is used as a general monitor of charge heterogeneity for golimumaband a different assay is used as the primary assay to monitordeamidation.

TABLE 9 Predicted Identities for Peaks Observed in cIEF Electropherogramof FB^(a) cIEF Peak Identity of Major Species Identity of Minor SpeciesB 2 HC Asp43, 2 SA HC Asp43, HC isoAsp43, 3 SA 2 HC isoAsp43, 4 SA C 2HC Asp43, 1 SA HC Asp43, HC isoAsp43, 2 SA 2 HC isoAsp43, 3 SA 1 2 HCAsp43 HC Asp43, HC isoAsp43, 1 SA 2 HC isoAsp43, 2 SA 2 HC Asp43, HCisoAsp43 2 HC isoAsp43, 1 SA HC Asp43, HC Asn43 3 2 HC isoAsp43 HCisoAsp43, HC Asn43 ^(a)SA is sialic acid; variants containing HC Asn43in place of Asp43 or isoAsp43 are also present at low levels but are notincluded in the table.

Introduction to Manufacturing Control Strategies

During large-scale commercial production, manufacturing controlstrategies are developed to maintain consistent drug substance (DS) anddrug product (DP) characteristics of therapeutic proteins (e.g.,therapeutic antibodies like golimumab), with regard to oligosaccharideprofile, bioactivity (potency), and/or other characteristics of the DSand DP (e.g., See characteristics identified in Table 10 and Table 11).For example, golimumab glycosylation is monitored as an in-processcontrol for formulated bulk (FB) at Stage 9 of the manufacturingprocess, with upper and lower specifications in place for mean % totalneutral oligosaccharides, % total charged oligosaccharides, and %individual neutral oligosaccharide species, G0F, G1F, and G2F.

TABLE 10 Representative comparison of selected golimumab characteristicsexpressed in Sp2/0 cells and CHO cells Test Parameter Sp2/0 Cells CHOCells DW-SE-HPLC % Purity 99.81% 99.61% % Aggregate  0.18%  0.39% %Fragment <LOD <LOD cSDS Reduced % Purity  98.6% 97.7% cSDS Non-Reduced %Purity  98.9% 96.8% Bioactivity % Bioactivity NA     83%^(a) cIEF peak 1   22%    28% peak 2    38%    37% peak 3    29%    21% peak C     8%    9% peak B     2%     4% Sum of major peaks    97%    95% Peptidemapping HC N393 deamidation   7.8%   9.3% HC N43 deamidation    67%   81% HC N43 Isomerization    25%    26% HC M261 oxidation <LOD   2.5%IMW mass NA all within 42 Daltons^(a) ^(a)Compared to reference material(expressed in Sp2/0 cells) <LOD - below limit of detection NA - NotApplicable for reference material produced in Sp2/0 cells

As shown in Table 10, there are differences in some of the selectedcharacteristics for golimumab produced in Sp2/0 cells and CHO cells.However, it was previously determined that differences in deamidationprofiles (e.g., levels of HC Asn43 deamidation) reflected in the cIEFand peptide mapping results are primarily caused by differences inprocessing (e.g., purification and/or storage) of the protein.Similarly, differences in bioactivity have been shown to be caused bydifferences in processing (i.e., large-scale commercial production vs.small-scale production) and are not considered to be caused by thedifference in the host cell used for expressing the protein.Furthermore, the bioactivity of golimumab expressed in CHO cells andpurified at small-scale is still within specification.

Oligosaccharide Profile of Golimumab

Golimumab is N-glycosylated at a single site on each heavy chain, onasparagine 306. These N-linked oligosaccharide structures can be any ina group of biantennary oligosaccharide structures linked to the proteinthrough the primary amine of the asparagine residue, but on golimumabthey consist primarily of biantennal core-fucosylated species, withgalactose and sialic acid heterogeneity. Individual oligosaccharidespecies include “G0F”, an asialo, agalacto core-fucosylated biantennaryglycan, “G1F”, an asialo, mono-galacto core-fucosylated biantennaryglycan, and “G2F”, an asialo, di-galacto core-fucosylated biantennaryglycan. Golimumab glycosylation is monitored as an in-process controlduring Stage 9 of manufacturing, with specifications in place for totalneutral oligosaccharides, total charged oligosaccharides, and individualneutral oligosaccharide species G0F, G1F, and G2F. A diagrammaticoverview of some of the primary N-linked oligosaccharide species ingolimumab IgG is shown in FIG. 24. The role of some of the enzymes inthe glycosylation maturation process, including roles of some divalentcations (e.g. Mn²⁺ and Cu²⁺) in these enzymatic processes are alsoshown.

Controlling Oligosaccharide Profile

Controlling the oligosaccharide profile of therapeutic antibodies iscritical, because changes in the oligosaccharide profile of arecombinant monoclonal antibody can significantly affect antibodybiological functions. For example, biological studies have shown thatthe distribution of different glycoforms on the Fc region cansignificantly impact antibody efficacy, stability, and effector function(J. Biosci. Bioeng. 2014 117(5):639-644; Bio-Process Int. 2011,9(6):48-53; Nat. Rev. Immunol. 2010, 10(5):345-352). In particular,afucosylation Mol. Biol. 368:767-779) and galactosylation (BiotechnotProg. 21:1644-1652) can play a huge role in the antibody-dependentcell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC), two important mechanisms by which antibodies mediate killingtarget cells through the immune function. In addition, high mannoselevels have been shown to adversely affect efficacy by increasingclearance of the antibody (Glycobiology. 2011, 21(7):949-959) and sialicacid content can affect anti-inflammatory activity (Antibodies. 20132(3):392-414). As a result of these biological consequences from changesin the oligosaccharide profile, regulatory agencies require control ofthe antibody glycosylation pattern to ensure adherence to lot releasespecifications for a consistent, safe and effective product.

Oligosaccharide Profile—Effects from Expression in Different Cells

Two commonly used host cell lines for the recombinant expression ofantibodies are Chinese hamster ovary cells (CHO) and mouse myeloma cells(e.g., Sp2/0 cells). CHO cells express recombinant antibodies which canbe virtually devoid of sialic acid glycan and the glycans can be up to99% fucosylated. In contrast, mouse myeloma cells express recombinantantibodies that can contain up to 50% sialic acid and generally haveless fucose. These differences can have significant effects on antibodyactivity in vivo, e.g., it has been shown that such differences canaffect the structure of the Fc-portion of the molecule and thereby alterantibody effector functions such as antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (see,e.g., U.S. Pat. No. 8,975,040). For example, reduced ADCC activity hasbeen noted with increased sialylated (charged) Fc glycans (Scallon etal. Mol Immunol 2007; 44:1524-34).

The effects on ADCC activity may be particularly important for anti-TNFantibodies because it has been suggested that for some approvedindications a crucial mode of action (MOA) for anti-TNF antibodies maybe destruction of TNF-α producing cells through ADCC, e.g., for treatinginflammatory bowel disease (IBD), such as Crohn's disease (CD) and/orulcerative colitis (UC). It has also been shown that Flixabi, a CHOderived biosimilar of Remicade (infliximab), has a higher mean ADCCactivity in a NK92-CD16a cell line. Infliximab is produced in Sp2/0cells and has an oligosaccharide profile that has a higher percentage ofcharged glycan than Flixabi that is produced in CHO cells (Lee et al.MAbs. 2017 August; 9(6): 968-977). Accordingly, it may be desirable toreduce sialylation and thus the percentage of charged glycans associatedwith anti-TNF antibodies.

In addition, antibodies produced in CHO and Sp2/0 cells can havesignificant differences in the levels of two glycan epitopes,galactose-α-1,3-galactose (α-gal) and the sialylated N-glycanNeu5Gc-α-2-6-galactose (Neu5Gc). For example, it has been shown that CHOcells can express antibodies with undetectable or only trace levels ofα-Gal and Neu5Gc, while Sp2/0 cells can express much higher levels ofthe two glycan structures (Yu et al., Sci Rep. 2016 Jan. 29; 7:20029).In contrast, humans are genetically deficient in the gene forbiosynthesizing α-gal and the gene responsible for production of Neu5Gcis irreversibly mutated in all humans. As a result, α-Gal and Neu5Gc arenot produced in humans. Furthermore, the presence of these non-humanglycan epitopes on therapeutic antibodies can cause undesirable immunereactions in certain human populations because of higher levels ofpre-existing antibodies to α-Gal and Neu5Gc. For example, anti-α-gal IgEmediated anaphylactic responses have been reported for Cetuximab (Chung,C. H. et al., N Engl J Med. 2008 Mar. 13; 358(11):1109-17) and thepresence of circulating anti-Neu5Gc antibodies has been reported topromote clearance of Cetuximab (Ghaderi et al., Nat Biotechnol. 2010August; 28(8):863-7).

Oligosaccharide of Golimumab Expressed in Sp2/0 Cells and CHO Cells

Compiled HPLC data from multiple commercial production runs of golimumabshowed that DS or DP produced in Sp2/0 cells comprises anti-TNFantibodies comprising total neutral oligosaccharide species ≥82.0% to≤94.4%, total charged oligosaccharide species ≥5.6% to ≤1 18.0%, andindividual neutral oligosaccharide species G0F ≥25.6% to ≤42.2%, G1F≥31.2% to ≤43.6%, and G2F ≥5.6% to ≤14.2%. As shown in Table 11 and FIG.25, the oligosaccharide profile of a golimumab expressed in CHO cells isdramatically different from golimumab expressed in Sp2/0 cells. Comparedto golimumab produced in Sp2/0 cells, the oligosaccharide profile forgolimumab produced in CHO cells is shifted toward very low levels ofcharged glycans and higher levels of neutral glycans that arepredominantly G0F. The oligosaccharide profile for golimumab produced inCHO cells comprises total neutral oligosaccharide species >99.0%, totalcharged oligosaccharide species <1.0%, and individual neutraloligosaccharide species G0F >60.0%, G1F <20.0%, and G2F <5.0%.Furthermore, no disialylated glycan species were detected by IRMA or byHPLC for golimumab produced in CHO cells and monosialylated glycanspecies were at very low levels based on HPLC analysis and undetectableby IRMA analysis.

TABLE 11 Representative results for IRMA and HPLC analysis of totalneutral, total charged, and other selected oligosaccharide species forgolimumab produced in Sp2/0 cells and CHO cells IRMA HPLC Glycans Sp2/0CHO Sp2/0 CHO G0F 28.9 67.4 32.2 82.0 G1F 33.1 11.6 36.0 11.9 G2F 10.3<LOD 10.0 3.4 Other neutral 13.5 21.0 7.3 2.2 Total neutral 85.7 100.0%85.5 >99.6 Monosialylated 13.1 <LOD 13.8 <1.0 Disialylated 1.2 <LOD <1.0<LOD Total charged 14.3 <LOD 14.5 <1.0 <LOD - below limit of detectionNumbers are % of totals

TABLE 12 Representative results for IRMA analysis of individualoligosaccharide species for golimumab produced in Sp2/0 cells and CHOcells Glycan Sp2/0 CHO G0F 28.9 67.4 G1F 33.1 11.6 G2F 10.3 <LOD Man51.7 2.2 G0 2.4 15.7 G1FS 5.0 <LOD *G1F-G1cNAc +Lys 0.4 <LOD *H5N3 +Lys1.3 0.7 *G1 +Lys 1.8 <LOD *H5N3F1 +Lys 0.3 <LOD *G2 +Lys 0.5 <LOD *G2FS−Lys 4.5 <LOD *H6N4F1S1 −Lys 3.6 <LOD *G2FS2 −Lys 1.2 <LOD *H6N4F1 −Lys2.4 <LOD *H7N4F1 −Lys 1.7 <LOD **G1F-G1cNAc −Lys <LOD <LOD **H5N3 −Lys<LOD <LOD **G1 −Lys 0.9 2.3 **H5N3F1 −Lys <LOD <LOD **H5N4 −Lys <LOD<LOD <LOD - below limit of detection Numbers are % of totals

Conclusion

Thus, as described supra, manufacturing control strategies are developedto maintain consistent drug substance (DS) and drug product (DP)characteristics of therapeutic proteins with regard to oligosaccharideprofile and/or other characteristics of the DS or DP (e.g., DS and/or DPcomprising the therapeutic antibody golimumab). In particular,controlling the oligosaccharide profile of therapeutic antibodies iscritical because changes in the oligosaccharide profile cansignificantly affect antibody biological functions. A point of controlfor the oligosaccharide profile of therapeutic antibodies is theselection of the cellular host for expression of the therapeuticantibodies. As presented herein, golimumab expressed in Sp2/0 cellscomprises anti-TNF antibodies having a heavy chain (HC) comprising anamino acid sequence of SEQ ID NO:36 and a light chain (LC) comprising anamino acid sequence of SEQ ID NO:37, wherein the oligosaccharide profileof the anti-TNF antibodies comprises total neutral oligosaccharidespecies ≥82.0% to ≤94.4%, total charged oligosaccharide species ≥5.6% to≤18.0%, and individual neutral oligosaccharide species G0F ≥25.6% to≤42.2%, G1F ≥31.2% to ≤43.6%, and G2F ≥5.6% to ≤14.2%.

In contrast, golimumab expressed in CHO cells comprises mammaliananti-TNF antibodies having a heavy chain (HC) comprising an amino acidsequence of SEQ ID NO:36 and a light chain (LC) comprising an amino acidsequence of SEQ ID NO:37, wherein the oligosaccharide profile of theanti-TNF antibodies comprises total neutral oligosaccharidespecies >99.0%, total charged oligosaccharide species <1.0%, andindividual neutral oligosaccharide species G0F >60.0%, G1F <20.0%, andG2F <5.0%. Furthermore, no disialylated glycan species were detected byIRMA or by HPLC for golimumab produced in CHO cells and monosialylatedglycan species were at very low levels based on HPLC analysis andundetectable by IRMA analysis.

These changes in the oligosaccharide profile for golimumab produced inCHO cells may provide a variety of benefits in different therapeuticindications and in different patient populations. For example, reducedsialylated (charged) Fc glycans on the anti-TNF antibodies may increaseADCC activity in vivo, such that efficacy is enhanced in the treatmentof certain diseases, e.g., inflammatory bowel disease (IBD), such asCrohn's disease (CD) and/or ulcerative colitis (UC). In addition, thereduction in sialylated species generally and the reduction of Neu5Gcspecifically for anti-TNF antibodies produced in CHO cells may provide abenefit by reducing undesirable immunogenic responses when administeredto humans. For example, reduced levels of Neu5Gc could reduce clearanceso that anti-TNF antibodies produced in CHO cells would have a longerhalf-life compared to anti-TNF antibodies expressed in Sp2/0 cells,especially for patient populations with higher levels of anti-Neu5Gcantibodies.

1. Anti-TNF antibodies comprising: (i) a heavy chain comprising an aminoacid sequence of SEQ ID NO:36; and (ii) a light chain comprising anamino acid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areexpressed in Chinese Hamster Ovary cells (CHO cells).
 2. The anti-TNFantibodies of claim 1, wherein the oligosaccharide profile of theanti-TNF antibodies comprises total neutral oligosaccharidespecies >99.0% and total charged oligosaccharide species <1.0%.
 3. Theanti-TNF antibodies of claim 1, wherein the oligosaccharide profile ofthe anti-TNF antibodies further comprises individual neutraloligosaccharide species G0F >60.0%, G1F <20.0%, and G2F <5.0%.
 4. Theanti-TNF antibodies of claim 1, wherein the anti-TNF antibodies have nodisialylated glycan species as determined by High Performance LiquidChromatography (HPLC) or Reduced Mass Analysis (RMA).
 5. The anti-TNFantibodies of claim 1, wherein the anti-TNF antibodies have a longerhalf-life or increased antibody-dependent cell-mediated cytotoxicity(ADCC) compared to anti-TNF antibodies expressed in Sp2/0 cells.
 6. Theanti-TNF antibodies of claim 5, wherein the anti-TNF antibodies are afollow-on biologic.
 7. A method of manufacture for producing anti-TNFantibodies comprising: (i) a heavy chain comprising an amino acidsequence of SEQ ID NO:36; and (ii) a light chain comprising an aminoacid sequence of SEQ ID NO:37, wherein the anti-TNF antibodies areproduced by a method of manufacture comprising: a. culturing ChineseHamster Ovary cells (CHO cells) with nucleotides encoding the anti-TNFantibodies; b. expressing the anti-TNF antibodies in the CHO cells; and,c. purifying the anti-TNF antibodies.
 8. The method of manufacture ofclaim 7, wherein the oligosaccharide profile of the anti-TNF antibodiescomprises total neutral oligosaccharide species >99.0% and total chargedoligosaccharide species <1.0%, and
 9. The method of manufacture of claim7, wherein the oligosaccharide profile of the anti-TNF antibodiesfurther comprises individual neutral oligosaccharide species G0F >60.0%,G1F <20.0%, and G2F <5.0%.
 10. The method of manufacture of claim 7,wherein the anti-TNF antibodies have no disialylated glycan species asdetermined by High Performance Liquid Chromatography (HPLC) or ReducedMass Analysis (RMA).
 11. The method of manufacture of claim 7, whereinthe anti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells.
 12. The method ofmanufacture of claim 11, wherein the anti-TNF antibodies are a follow-onbiologic.
 13. A composition comprising anti-TNF antibodies: (i) a heavychain comprising an amino acid sequence of SEQ ID NO:36; and (ii) alight chain comprising an amino acid sequence of SEQ ID NO:37, whereinthe anti-TNF antibodies are expressed in Chinese Hamster Ovary cells(CHO cells).
 14. The composition of claim 13, wherein theoligosaccharide profile of the anti-TNF antibodies comprises totalneutral oligosaccharide species >99.0% and total charged oligosaccharidespecies <1.0%.
 15. The composition of claim 13, wherein theoligosaccharide profile of the anti-TNF antibodies further comprisesindividual neutral oligosaccharide species G0F >60.0%, G1F <20.0%, andG2F <5.0%.
 16. The composition of claim 13, wherein the anti-TNFantibodies have no disialylated glycan species as determined by HighPerformance Liquid Chromatography (HPLC).
 17. The composition of claim13, wherein the anti-TNF antibodies have a longer half-life or increasedantibody-dependent cell-mediated cytotoxicity (ADCC) compared toanti-TNF antibodies expressed in Sp2/0 cells.
 18. The composition ofclaim 17, wherein the anti-TNF antibodies are a follow-on biologic.